SIRNAS TARGETING REGULATION OF LPA GENE EXPRESSION AND USES THEREOF

Abstract

The present disclosure provides siRNAs targeting the regulation of LPA gene expression and uses thereof. Cell experiment results show that the oligonucleotide duplexes of the present disclosure can significantly inhibit the expression of LPA genes, 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 biological medicine, and particularly relates to siRNA targeting the regulation of LPA gene expression and use thereof. BACKGROUND

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

[0003] In terms of cardiovascular diseases, high levels of LPA gene expression are closely related to the occurrence and development of atherosclerosis and the increase of thrombosis risk. Apolipoprotein A (Apo(a)) can promote the aggregation and oxidation of lipoprotein particles, thereby leading to impaired vascular endothelial cell function, inflammatory cell infiltration, and the formation and instability of atherosclerotic plaques. In addition, LPA gene polymorphism is also associated with the susceptibility of cardiovascular diseases such as coronary heart disease and myocardial infarction, which further highlights the potential value of regulating LPA gene expression in the prevention and treatment of cardiovascular diseases.

[0004] Currently, the treatment methods for regulating LPA gene expression are relatively limited. Traditional drugs for treating cardiovascular diseases and metabolic diseases mainly act through regulating blood lipids, anti-inflammatory, anti-platelet aggregation and other pathways, but these drugs often cannot directly target LPA gene, and the treatment effect has certain limitations, and may be accompanied by more side effects. Therefore, it is of great clinical significance and market prospect to develop a new method that can specifically and efficiently regulate the expression of LPA gene.

[0005] Oligonucleotides are short DNA or RNA molecules, oligomers, which can bind to their complementary oligonucleotides, DNA or RNA in a sequence-specific manner to form duplexes, or, although less common, in some cases to form higher-order hybrids. Oligonucleotides can bind to complementary RNA strands in a sequence-specific manner, and after hybridization, they can induce RNase H to cleave the target RNA, thereby reducing the expression of the target gene. In addition, in natural oligonucleotides, nucleotides are connected by phosphodiester bonds, and under physiological conditions, oligonucleotides are particularly sensitive to nucleases, so when preparing oligonucleotide drugs, natural, unmodified or unmodified oligonucleotides are easily degraded in vivo, and thus have very limited activity, and therefore have poor drug properties. Modifying oligonucleotides is an effective way to improve their activity, which can improve their stability to nucleases, affinity to RNA, and better promote cellular endocytosis and tissue targeting, thereby effectively regulating the expression of target genes.

[0006] Currently, oligonucleotides, especially siRNA, which can effectively inhibit the expression of LPA gene, need to be further explored and improved. SUMMARY

[0007] To solve the technical problem of lack of siRNA duplexes for effectively targeting and regulating the expression of LPA gene in the prior art, the present disclosure provides siRNA duplexes for targeting and regulating the expression of LPA gene and their use in preventing and treating cardiovascular and cerebrovascular diseases. The present disclosure screens a plurality of siRNA modifiers having a significant inhibitory effect on the expression of LPA gene by modifying the basic sequence of siRNA, and provides corresponding siRNA conjugates.

[0008] The technical solutions of the present disclosure include but are not limited to:

[0009] In one aspect, the present disclosure provides an siRNA duplex, which comprises an oligonucleotide duplex composed of a sense strand and an antisense strand.

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

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

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

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

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

[0015] In some embodiments, the recombinant cell is selected from a sulfur-oxidizing Rhodopseudomonas and a ribonuclease III-deficient Corynebacterium glutamicum.

[0016] In another aspect, the present disclosure provides a method for preparing an siRNA duplex, which comprises culturing the above-mentioned recombinant cell or chemical synthesis.

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

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

[0019] In some embodiments, the method is 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 use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid-protein complex, or the above-mentioned pharmaceutical composition in the manufacture of a medicament for treating a disease associated with LPA gene expression.

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

[0023] In some embodiments, the disease associated with LPA gene expression is selected from the group consisting of Berger’s disease, peripheral arterial disease, coronary arterial disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery obstruction, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery obstruction, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis, and any other disease associated with elevated levels of Lp(a) particles, and other yet unidentified related conditions, pathologies or syndromes.

[0024] 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 use in treating a disease 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 to a subject in need thereof an effective amount of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid-protein complex, or the above-mentioned pharmaceutical composition.

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

[0027] In another aspect, the present disclosure provides a use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid-protein complex, or the above-mentioned pharmaceutical composition for the preparation of a medicament 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 to a subject in need thereof an effective amount of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid-protein complex, or the above-mentioned pharmaceutical composition.

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

[0030] To make the present disclosure more readily understood, certain terms are defined first. Additionally, it should be noted that whenever a value or a range of values for a parameter are recited, it is intended that the intervening values between the upper and lower limits are also intended to be encompassed unless otherwise indicated.

[0031] The articles "a" and "an" as used herein mean one or more than one (i.e., at least one) of the grammatical article with the understanding that when there is more than one, the elements are the same unless otherwise indicated.

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

[0033] The term "or" as used herein is intended to mean "and / or" unless otherwise indicated.

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

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

[0036] The term "LPA gene" can be a wild-type LPA gene, or a mutant of the LPA gene that has sequence variations. Numerous sequence variations in the LPA gene have been identified and can be found, for example, in the NCBI dbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov / snp).

[0037] "G," "C," "A," and "U" each generally represent a nucleotide comprising, respectively, guanine, cytosine, adenine, and uracil as the base. "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 will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide (as described further below) or an alternative substituent moiety. The skilled artisan will be 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 substituent 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 a nucleotide sequence of the disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such substituent moieties are suitable for use in, including but not limited to, the double-stranded ribonucleic acids, double-stranded ribonucleic acid modifications, double-stranded ribonucleic acid conjugates, pharmaceutical compositions, and methods of the disclosure, among others.

[0038] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein to refer to base pairing between the sense strand and the antisense strand 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 aspects herein, a first nucleotide sequence can be 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% degree of sequence complementarity with the second nucleotide sequence. In an exemplary embodiment, 18 of 20 nucleobases of the first nucleotide sequence pair with the corresponding region of the second nucleotide sequence, achieving 90% complementarity.

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

[0040] As is well known in the art, the term "siRNA duplex," "double-stranded RNAi agent," "RNAi agent," "small interfering ribonucleic acid," or "siRNA" refers to a small interfering ribonucleic acid RNAi molecule. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silencing RNA. The siRNA typically comprises a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand), each strand being 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 a target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a duplex or duplex region. The sense and antisense strands of the siRNA can form a blunt-end 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 product of Dicer production, which can form a RISC substrate in vivo. Efficient extensions of the Dicer substrate have been described in US 8349809 and US 8513207, incorporated herein by reference. In some embodiments, both the sense and antisense strands have a 3' overhang 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] Also, in the present context, "siRNA" in some cases also refers to "base sequence". In the present context, "base sequence" in some cases specifically refers to an siRNA duplex in which each nucleotide in the double-stranded ribonucleic acid is an unmodified nucleotide, also appearing throughout the text as "motif", "siRNA motif", etc. Thus, in the present context, "siRNA", "base sequence", "motif", "siRNA motif" can be used interchangeably, and their meaning also includes the corresponding nucleotide arrangement order of the referred siRNA duplex. In the present context, the skilled person can clearly understand the exact technical meaning referred to by them according to the technical meaning of the context. In addition, the 5' terminal nucleotide of the antisense strand of the motif can be linked with a 5' phosphate group or a 5' phosphate derivative group or can not be linked with a 5' phosphate group or a 5' phosphate derivative group.

[0042] In the present context, "siRNA modifier" refers to a double-stranded ribonucleic acid comprising at least one modified nucleotide, in some cases also appearing as "double-stranded ribonucleic acid modifier". In the present context, different modifications of the siRNA motif are made to prepare the corresponding siRNA modifier. For example, in some embodiments, the motif is modified using an alternating modification to obtain an alternating modified siRNA modifier. In other embodiments, the motif is modified using a specific modification template modification to obtain a specific modification template modified siRNA modifier. In yet other embodiments, the motif is modified using an off-target prevention modification in the present context to obtain an off-target prevention modified siRNA modifier. In some cases, multiple different modifications can be used to modify the same siRNA motif to obtain the corresponding siRNA modifier with multiple modifications.

[0043] In the present context, "siRNA conjugate" refers to a conjugate of a double-stranded ribonucleic acid conjugate or a double-stranded ribonucleic acid modifier obtained by linking a conjugate group to the double-stranded ribonucleic acid, the double-stranded ribonucleic acid modifier. Preferably, "siRNA conjugate" refers to a conjugate of a double-stranded ribonucleic acid modifier.

[0044] In some cases in the present context, "siRNA" not only refers to the unmodified siRNA duplex (or siRNA motif) described above, but can also refer to the corresponding siRNA modifier and / or siRNA conjugate, for example, in the context of including but not limited to therapeutic methods, therapeutic agents, etc., siRNA refers to at least one of siRNA motif, siRNA modifier and / or siRNA conjugate. For the skilled person, the specific technical meaning referred to by them can be clearly understood in combination with the context.

[0045] The term "antisense strand" refers to the strand of a double-stranded ribonucleic acid (e.g., an RNA duplex herein) that includes a region of substantial complementarity 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 perfectly complementary to the target sequence, mismatches can be internal or at the terminal regions of the molecule. Generally, the most tolerated mismatches are at the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' terminus. The target sequence is, for example, derived from Homo sapiens lipoprotein(a) (LPA), mRNA with accession number 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 of substantial complementarity to a region of an antisense strand as that term is defined herein.

[0047] The term "nonsense sequence" is a nucleic acid sequence that is artificially designed or selected to not bind significantly complementary to the mRNA of any known functional gene in the target organism, and thus theoretically cannot cause specific gene silencing. This sequence serves as an experimental control to exclude the influence of non-targeting factors (e.g., transfection stress, vector backbone effects, or innate immune responses) on the results in the RNA interference experiment.

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

[0049] A "double-stranded region" as used herein means a region of a double-stranded ribonucleic acid in which the sense strand and the antisense strand are complementary or substantially complementary by base pairing, and which is typically 19, 20, 21 bases in length, and in some cases can be other than 19, 20, 21 bases in length. Also, one of skill in the art will readily appreciate from the conventions of the art that a length of 19, 20, 21 bases can also be referred to as a length of 19, 20, 21 base pairs (bp).

[0050] Accordingly, it will be understood by those skilled in the art that, for a double-stranded ribonucleic acid comprising overhangs, the structure can be such that the overhangs are comprised at the 5' end of the sense strand of the double-stranded region, or at the 3' end of the sense strand of the double-stranded region, or at both ends of the double-stranded region; and for a double-stranded ribonucleic acid not comprising overhangs, i.e. a "blunt-ended" double-stranded ribonucleic acid, the entire length thereof is a double-stranded region, as described hereinabove.

[0051] It will be readily understood by those skilled in the art that the definitions described hereinabove with respect to "overhang", "blunt end", "double-stranded region", etc. also apply to double-stranded ribonucleic acid modifications, double-stranded ribonucleic acid conjugates.

[0052] The terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art, i.e. in a double-stranded ribonucleic acid molecule, the bases of one strand pair with the bases of 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 comprises one purine and one pyrimidine. When the adenine on one strand always pairs with the thymine (or uracil) on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be deduced from the sequence of its complementary strand. Accordingly, "mismatch" means that the bases at the 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 mismatches between the two nucleotide strands in question, i.e. there are 1, 2 or 3 mismatches between the two nucleotide strands in question.

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

[0055] As used herein, "difference" refers to the presence of a different nucleotide in the nucleotide sequence of a ribonucleic acid strand than the nucleotide sequence of the ribonucleic acid strand, e.g. when it is stated that a sense strand and / or an antisense strand differs from a particular sequence by 1-3 nucleotides, it is meant that the sense strand and / or the antisense strand differs from the ribonucleic acid strand as set forth in the particular sequence by 1-3 different nucleotides.

[0056] The term "alternating modification" or "oxyfluoro alternating modification" refers to the modification of each nucleotide in the order of nucleotide sequence of each strand of the double-stranded ribonucleic acid with 2'-methoxy (2'-OMe) modification for odd-numbered positions and 2'-fluoro (2'-F) modification for even-numbered positions. For example, for the antisense strand of an siRNA, the odd-numbered positions (i.e., positions 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23) are modified with 2'-methoxy and the even-numbered positions (i.e., positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22) are modified with 2'-fluoro. For example, for the sense strand, the nucleotides on the sense strand that pair with the 2'-methoxy modified nucleotides of the antisense strand are modified with 2'-fluoro and the nucleotides on the sense strand that pair with the 2'-fluoro modified nucleotides of the antisense strand are modified with 2'-methoxy. In some embodiments, the nucleotides at the odd-numbered positions of the sense strand are 2'-fluoro modified nucleotides and the nucleotides at the even-numbered positions of the antisense strand are 2'-methoxy modified nucleotides. In some embodiments, the 5' end of the sense strand of an "alternating modification" or "oxyfluoro alternating modification" double-stranded ribonucleic acid modification has 3', 5'-phosphorothioate linkages between the first and second nucleotides and between the second and third nucleotides. The 5' end of the antisense strand of an "alternating modification" or "oxyfluoro alternating modification" double-stranded ribonucleic acid modification has 3', 5'-phosphorothioate linkages between the first and second nucleotides and between the second and third nucleotides and the 3' end of the antisense strand of an "alternating modification" or "oxyfluoro alternating modification" double-stranded ribonucleic acid modification has 3', 5'-phosphorothioate linkages between the first and second nucleotides and between the second and third nucleotides.

[0057] For RNA interference (RNAi), the inhibition of a target gene is achieved by the AGO2 protein loading the antisense strand of an siRNA and forming a silencing complex (RISC) that cleaves the mRNA transcript of the gene. The loading of the silencing complex (RISC) requires the 5' end of the antisense strand to be phosphorylated (5'-phosphate). The phosphorylation of the 5' end can occur naturally in the cell by the cleavage and polyadenylation factor I subunit 1 (Clp1) or it can be achieved by chemical synthesis. The term "natural 5' end phosphorylation" or "simple direct 5' end phosphorylation" refers to the phosphorylation of the 5' end of the antisense strand of an siRNA being completed in the cellular environment and not by chemical synthesis.

[0058] In the present context, a "conjugate group" is a GalNAc derivative attached to an oligonucleotide. In some cases, a conjugate group comprises a targeting group (also referred to as a ligand), optionally further comprising a linker, e.g. a GalNAc derivative linked to an oligonucleotide via a linker (e.g. a divalent, trivalent or tetravalent branched linking arm), and also e.g. a GalNAc derivative attached to an oligonucleotide via a monovalent linking arm. In most cases, both "ligand" and "conjugate group" have the meaning well known in the art.

[0059] The term "inhibit", as used herein, can be used interchangeably with "reduce", "silence", "down-regulate", "suppress" and other similar terms, and includes inhibition at any level. In the present context, "inhibit" in some cases refers to the meaning "reduce", the specific meaning referred to by the skilled person being clear from the context.

[0060] As used herein, the phrase "inhibit expression of LPA" includes inhibiting expression of any LPA gene (such as, e.g., 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 a transgenic LPA gene in the context of a genetically manipulated cell, cell population, or organism.

[0061] "Inhibit expression of a LPA gene" includes inhibition of a LPA gene at any level, e.g. at least partial inhibition of expression of a LPA gene, 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] Expression of a LPA gene can be assessed based on any variable level associated with LPA gene expression, such as LPA mRNA level or LPA protein level. 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 utilized in the art, such as a pre-dose baseline level or a level determined from a similar subject, cell, or sample that was not treated or treated with a control (e.g. a buffer control or an inert agent control).

[0063] In this document, in some cases, the meaning of "modulate" can be the same as "inhibit"; accordingly, "modulating LPA gene expression" can mean "inhibiting LPA gene expression". The specific technical meaning will be clear to one of skill in the art in view of the context.

[0064] As used herein, "patient" or "subject" is intended to include a human or non-human animal, preferably a mammal, such as a monkey. More preferably, the subject or patient is a human.

[0065] As used herein, "LPA-related disease" is intended to include any disease associated with LPA gene or protein. Such disease can be caused, for example, by an excess production of LPA protein, by a mutation of LPA gene, by an abnormal cleavage of LPA protein, by an abnormal interaction between LPA and other proteins or other endogenous or exogenous substances. Exemplary LPA-related diseases include Buerger's disease, peripheral arterial disease, coronary artery disease, metabolic syndrome, acute coronary syndrome, aortic valve stenosis, aortic valve regurgitation, aortic dissection, retinal artery occlusion, cerebrovascular disease, mesenteric ischemia, superior mesenteric artery occlusion, renal artery stenosis, stable / unstable angina, acute coronary syndrome, heterozygous or homozygous familial hypercholesterolemia, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis, stroke, atherosclerosis, thrombosis, coronary heart disease or aortic valve stenosis and / or any other disease associated with an elevated level of Lp(a) particles as well as other yet unidentified related conditions, pathologies or syndromes.

[0066] As used herein, "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient for treating an LPA-related disease, is sufficient to effect treatment (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by LPA expression, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0067] As used herein, a "prophylactically effective amount" means an amount of an RNAi agent sufficient to prevent or ameliorate a disease or one or more symptoms of a disease, including when administered to a subject who is not yet experiencing or displaying symptoms of a LPA-associated disease, but who is at risk for developing the disease. Ameliorating a disease includes slowing the progression of the disease or reducing the severity of the disease that follows. The "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the risk of the disease, and the subject's history, age, body weight, family history, genetic makeup, type of prior or concomitant treatments, if any, and other individual characteristics of the subject.

[0068] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents used in the methods of the 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 isolated from a subject, as well as a collection of fluids, cells or tissues present in a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from a tissue, organ or a localized region. For example, a sample can be derived from a particular organ, organ portion, or fluids or cells within these organs. In certain embodiments, a sample can be derived from a liver (e.g., the entire liver or certain segments of the liver, or certain types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject. In other embodiments, a "sample derived from a subject" refers to liver tissue (or sub-components thereof) derived from the subject.

[0070] In this document, when referring to any nucleotide position of any strand of an siRNA motif, siRNA modifier, siRNA conjugate, siRNA duplex, etc., the 5' to 3' direction is meant, unless otherwise specified.

[0071] In one aspect, the disclosure provides an siRNA duplex comprising a sense strand and an antisense strand forming a region of complementary base pairing, the antisense strand comprising a fragment of at least 15, 16, 17, 18, or 19 contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 122, 131, 148, 151, 152, 171, or a modified fragment thereof.

[0072] In some embodiments, the sense strand comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 13, 22, 39, 42, 43, 62, or a modification thereof.

[0073] In some embodiments, the reverse complementary duplex region is 17-21 bp in length, e.g., 20 or 21 bp.

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

[0075] In some embodiments, the siRNA duplex comprises any one or a combination of at least two of the following oligonucleotide duplexes, optionally each independently comprising at least one modified nucleotide:

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

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

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

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

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

[0081] (6) the sense strand has a sequence as set forth in SEQ ID NO: 62, or a fragment thereof, or a modified sequence of the sequence or fragment thereof; and the antisense strand has a sequence as set forth in SEQ ID NO: 171, or a fragment thereof, or a modified sequence of the sequence or 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 by 1-3 nucleotides from any one of SEQ ID NOs: 13, 22, 39, 42, 43, and 62.

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

[0085] In some embodiments, the sense strand has the same number of nucleotides as the antisense strand 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, each of the sense strand and the antisense strand independently comprises at least one modified nucleotide.

[0096] In some embodiments, the at least one modified nucleotide is selected from any one or a combination of at least two of the following: a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-0-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0097] In some embodiments, the modification of all nucleotides on the sense strand and the antisense strand is a modification of the 2' position of the ribose of the nucleotide.

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

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

[0100] In some embodiments, the modification of the 2' position of the ribose of each nucleotide is selected from an alternating combination of a 2'-methoxy modification and a 2'-fluoro modification.

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

[0102] In some embodiments, the nucleotides are connected to each other by 3',5'-phosphodiester linkages.

[0103] In some embodiments, the nucleotides are connected to each other by 3',5'-phosphorothioate diester linkages.

[0104] In some embodiments, the foregoing oligonucleotide has alternating fluoroxy modifications.

[0105] In some embodiments, the 3' terminal and / or 5' terminal 1st-2nd nucleotides of the sense strand and / or the antisense strand of the siRNA modification are linked by 3',5'- phosphorothioate diester bond, for example, in some embodiments, a chiral pure 3',5'- phosphorothioate diester bond is formed. In some embodiments, 1, 2, or 3 3',5'- phosphorothioate diester bonds can be contained between the 5' terminal starting 1st-4th nucleotides of the sense strand and / or between the 3' terminal starting 1st-4th nucleotides of the antisense strand. In some embodiments, both the 3',5'-phosphorothioate bonds are present between the 5' terminal starting 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the sense strand; both the 3',5'-phosphorothioate bonds are present between the 5' terminal starting 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the antisense strand, and both the 3',5'-phosphorothioate bonds are present between the 3' terminal starting 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the antisense strand.

[0106] In some embodiments of the present application, the modification pattern at the 2' position of each ribose nucleotide is: all odd positions of the sense strand are 2'-fluoro modified, and all even positions are 2'-methoxy modified; and all odd positions of the antisense strand are 2'-methoxy modified, and all even positions are 2'-fluoro modified.

[0107] Both the 3',5'-phosphorothioate bonds are present between the 5' terminal starting 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the sense strand; both the 3',5'-phosphorothioate bonds are present between the 5' terminal starting 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the antisense strand, and both the 3',5'-phosphorothioate bonds are present between the 3' terminal starting 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides of the antisense strand. The modified siRNA duplexes are, for example, A01501-AL, A02401-AL, A04101-AL, A04401-AL, A04501-AL, A06601-AL.

[0108] The present disclosure provides an siRNA conjugate comprising an siRNA duplex as described in the present disclosure and a conjugate group linked to the siRNA duplex.

[0109] The double-stranded ribonucleic acid, double-stranded ribonucleic acid modifications of the present disclosure can be optionally linked to one or more conjugate groups. The conjugate group can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the conjugate group can be linked to the sense strand. In preferred embodiments, the conjugate group is linked at the 3' end of the sense strand. In one embodiment, the conjugate group has any GalNAc structure.

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

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

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

[0113] In some embodiments, the pharmaceutically acceptable targeting group can be a conventional ligand in the field of siRNA administration, such as various ligands described in WO2009082607A2, incorporated 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 liver cell 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 liver cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a liver surface asialoglycoprotein receptor (ASGPR). The classes of such ligands are well known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of the target cell, mediating delivery of the siRNA linked to the ligand to the target cell.

[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, tetravalent. That is, after the siRNA molecule forms the siRNA conjugate with the conjugation 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 conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0116] The targeting group can be linked to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art according to the specific type of the targeting group. The linkers, the types of the targeting groups, and the ways of linking to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated by reference in its entirety.

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

[0118] ,

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

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

[0121] .

[0122] In some embodiments, the conjugation 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] R1is oxygen or sulfur;

[0127] R2is 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 -, wherein e is an integer from 0 to 7;

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

[0131] X1is -(CH2) f - or -(CH2CH2O) f CH2-, and f is an integer from 1 to 5;

[0132] X2is -(CH2) g -, and g is an integer from 1 to 6;

[0133] X3is oxygen or sulfur;

[0134] Y1is 0 or 1;

[0135] Y2is 0, 1, or 2;

[0136] X4is CH2when Y3is 1; CH when Y3is 2; and carbon when Y3is 3;

[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 conjugate of the present disclosure can have any of the following structures:

[0145] ,

[0146] ,

[0147] ,

[0148] , or

[0149] .

[0150] In some embodiments, the conjugate group has 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] R1is oxygen or sulfur;

[0155] R2is 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 - wherein e is an integer from 0 to 7;

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

[0159] X1is -(CH2) f - or -(CH2CH2O) f CH2- wherein f is an integer from 1 to 5;

[0160] X2is -(CH2) g - wherein g is an integer from 1 to 6;

[0161] X3is oxygen or sulfur;

[0162] Y1is 0 or 1;

[0163] Y2is 0, 1 or 2;

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

[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, the conjugate group has, for example, any of the following structures:

[0169]

[0170] In some embodiments, the siRNA conjugate of the disclosure has, for example, any of the following structures:

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] wherein either or both of the sense strand, the antisense strand, or both can be linked to a conjugate group G4, G5, G6, G7, G101, G102, G103, G105, or G106.

[0177] In some embodiments, the conjugate group is linked at the 3’ end of the sense strand.

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

[0179] ​​​​​​​​​​In the present disclosure, the term "nucleic acid protein complex" refers to the siRNA binding to Argonaute protein (AGO) to form an induced silencing complex (RISC). The siRNA is then unwound into a sense strand and an antisense strand. The sense strand is degraded and the antisense strand (guide strand) RISC binds to the target mRNA homologous to the siRNA through base pairing. RISC has the function of a nuclease, and siRNA guides RISC to cut the homologous single-stranded mRNA, resulting in the loss of function of mRNA, i.e. unable to translate to produce protein, that is, to make the gene "silence".

[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 the group consisting of a recombinant viroid-derived circular RNA vector, a tRNA, a rRNA scaffold, and a chimeric tRNA / pre-miRNA 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 the group consisting of a Sulfurospirillum and a ribonuclease 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 comprised in the nucleic acid molecule of the present application. A "recombinant cell" is a cell in which at least one recombinant vector has been introduced that can express the nucleic acid molecule or at least one strand of this nucleic acid molecule.

[0185] The present disclosure also provides a method of preparing the siRNA as described in the present disclosure, the method comprising culturing the aforementioned recombinant cell, or directly obtaining the siRNA using chemical synthesis and mixing.

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

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

[0188] The pharmaceutical compositions comprising the RNAi agents of the present disclosure can be, for example, a solution with or without a buffer or a composition containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micelle formulations, emulsions, and gene therapy vectors.

[0189] In the methods 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 buffered solution. The buffered solution can include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and the osmolality of the buffer containing the siRNA can be adjusted such that it is suitable for administration to a subject.

[0190] In some embodiments, the buffered solution further comprises an agent for controlling the osmolality of the solution such that the osmolality is maintained at a desired value, for example, at the physiological value of human plasma. Solutes that can be added to the buffered solution to control the 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 in a dosage sufficient to inhibit expression of an LPA gene. Generally, a suitable dosage of the siRNA of the present disclosure is in the range of about 0.001 to about 200.0 milligrams 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, the siRNA (e.g., siRNA conjugate) can be administered at 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, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg per single dose.

[0192] The pharmaceutical composition can be administered once a day, or multiple times at different intervals from 1 to 365 days, or the siRNA can be administered in two, three or more sub-doses at appropriate intervals throughout the year, or even continuously via a controlled release formulation using a continuous infusion or delivery. In this case, the siRNA contained in each sub-dose must be correspondingly less so as to achieve the total daily dose. Dose units can also be compounded for delivery over several days, for example using conventional sustained release formulations that provide a sustained release of siRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at a particular site, and can be used with the agents of the present disclosure. In this embodiment, the dose unit comprises a corresponding plurality of daily doses.

[0193] In other embodiments, a single dose of the pharmaceutical composition can be sustained for a long duration, 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 disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once a week. In other embodiments of the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once a month.

[0194] Those of skill in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and other existing diseases. In addition, treatment of a subject with a therapeutically effective dose of the composition can include a single treatment or a series of treatments. Effective doses and in vivo half-lives of the various siRNAs encompassed by the present disclosure can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0195] The pharmaceutical compositions of the disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., through a skin patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal; intranasal; epidermal and transdermal, oral or parenteral. Parenteral administration includes subcutaneous, intracutaneous, intradermal, intramuscular, intraperitoneal or intravenous injection or infusion; subdermal, e.g., via implantation devices; or intracranial, e.g., intracerebral, intrathecal or intraventricular, administration.

[0196] The siRNAs for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. 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), which has an external membrane formed of lipophilic material and an aqueous interior located inside. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not include the siRNA composition (although in some instances, it can). Liposomes are useful for transferring and delivering active ingredients to a site of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is administered to a tissue, the liposome bilayer fuses with the bilayer of a cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the siRNA, are delivered into the cell, where the siRNA can specifically bind to a target RNA and can mediate RNA interference (RNAi). In some cases, the liposomes are also specifically targeted, e.g., to direct the siRNA to a particular cell type.

[0197] Liposomes containing siRNA can be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed with the lipid components. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The siRNA preparation is then added to the micelles comprising the lipid components. The cationic groups on the lipids interact with the siRNA and condense around the siRNA to form liposomes. After condensation, the detergent is removed, e.g., by dialysis, to obtain the corresponding liposomal preparation of siRNA.

[0198] siRNAs, e.g., RNA duplexes of the disclosure, can be encapsulated in a lipid formulation (e.g., LNP or other nucleic acid-lipid particle).

[0199] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs contain a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., at sites physically separate from the site of administration).

[0200] In one embodiment, the mass ratio of the lipid to the 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 nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

[0202] In some preferred embodiments, the cationic lipid is a compound of structure (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C 6～15 linear alkyl; L2is C 12～25 branched alkyl. For example, YK-009 of structure (I-I), and the like (see patent CN114044741B, the entire contents of which are incorporated herein by reference, including therein the general formula and specific compounds, etc.).

[0203] (I)

[0204] (I-I)

[0205] In some preferred embodiments, the cationic lipid is a compound of structure (II), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 2~8 alkylene; G2is C 2~8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6~25 linear or branched alkyl; R2is C 6~25 linear or branched alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 of structure (II-I), YK-402 of structure (II-II), YK-407 of structure (II-III), and the like (see patent CN115784921B, the entire contents of which are incorporated herein by reference, including therein the general formula and specific compounds, etc.).

[0206] (II)

[0207] (II-I)

[0208] (II-II)

[0209] (II-III)

[0210] In some preferred embodiments, the cationic lipid is a compound of structure (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 1~6 alkylene; G2is C 2~8 alkylene; R1is C 6~20 linear or branched alkyl; R2is C 12~25 branched alkyl; G3is: 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 structure (III-I), YK-202 of structure (III-II), etc. (see patent CN115677518B, the entire contents of which are incorporated herein by reference, including therein the general formula and specific compounds, etc.).

[0211] (III)

[0212] (III-I)

[0213] (III-II)

[0214] In some preferred embodiments, the cationic lipid is a compound of structure (IV), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1~8 alkylene; G2is C 2~8 alkylene; R1is C 6~25 linear or branched alkyl; R2is C 12~25 linear or branched alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3is -CH3or -CH2CH3or -CH2CH2OH For example, YK-305 of the structure of Formula (IV-I), YK-310 of the structure of Formula (IV-II), and the like (see patent CN115745820B, the entire contents of which are incorporated herein by reference, including therein the general formulae and specific compounds, etc.).

[0215] (IV)

[0216] (IV-I)

[0217] (IV-II)

[0218] In some preferred embodiments, the cationic lipid is a compound of the structure of Formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G 1 and G 2 each independently is unsubstituted C6-C 10 alkylene; G 3 is unsubstituted C1-C 12 alkylene; R 1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; and R 5 is H or C1-C6 alkyl; for example, ALC0315 of the structure of Formula (V-I), and the like (see patent CN108368028B, the entire contents of which are incorporated herein by reference, including therein the general formulae and specific compounds, etc.).

[0219] (V)

[0220] (V-I)

[0221] In some preferred embodiments, the cationic lipid is a compound of the structure of Formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4is 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 structure (VI-I) (see patent application CN110520409A, the entire contents of which are incorporated herein by reference, including in particular the general formulae and specific compounds therein, etc.).

[0222] (VI)

[0223] (VI-I)

[0224] In some preferred embodiments, the cationic lipid is a compound of structure (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, including in particular the general formulae 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 group consisting of 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-didodecanoyl-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-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-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 16.0 PE), 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-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl- sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidyl ethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearophosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl- phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0234] In some preferred embodiments, the structural lipid is selected from any one or a combination of at least two of the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, a-tocopherol, corticosteroids.

[0235] In some preferred embodiments, the polymeric conjugated lipid is selected from any one or a combination of at least two of the group consisting of: 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 polymeric conjugated lipid is selected from any one or a combination of at least two of the group consisting of: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristyl glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bimatiryl 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 generated from a variety of components, examples of which include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. For example, preferred are formulations that target the liver when treating liver disorders, such as liver cancer.

[0238] Pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or fine solid carriers or both, and then, if necessary, shaping the product.

[0239] Compositions of the present disclosure can be formulated in any of a number of 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 comprise substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension can also comprise stabilizers.

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

[0241] A "pharmaceutical carrier" or "excipient" in contrast to a carrier compound, is a pharmaceutically acceptable solvent, suspending agent or other vehicle with which non- nucleic acid components are administered to an animal. The excipient can be liquid or solid and is selected with the planned manner of administering the particular pharmaceutical composition in mind and to provide for the most effective delivery of the nucleic acid. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oil, 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 that do not deleteriously react with the nucleic acid and are non-toxic to the animal can also be used to formulate compositions of the present disclosure for parenteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0243] Formulations for topical administration of nucleic acids can include sterile or non-sterile aqueous solutions, non-aqueous solutions, or nucleic acid solutions in liquid or solid oil bases. These solutions also can include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not cause toxic reactions with the nucleic acids can be used.

[0244] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0245] The present disclosure also provides methods for treating or preventing diseases and conditions that can be modulated by down-regulating LPA gene expression. For example, Buerger's disease, peripheral arterial 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, hyperapobetalipoproteinemia, 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 yet unidentified related conditions, pathologies or syndromes.

[0246] The siRNAs of the present disclosure can be administered to a subject using any mode of administration known in the art, including, but not limited to, subcutaneously, intravenously, intramuscularly, intraocularly, intrabronchially, intrapleurally, intraperitoneally, intraarterially, translymphatically, trans cerebrospinal, and any combination thereof. In preferred embodiments, the agents are administered subcutaneously.

[0247] In additional embodiments, the siRNA is administered in combination with an additional therapeutic agent. The siRNA and the additional therapeutic agent can be administered in combination in the same composition, e.g., 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 disorders. For example, additional therapeutic agents include: administering to the subject one or more siRNAs of the present application; administering to the subject a non-LPA RNAi therapeutic; and performing a behavior modification in the subject. In some embodiments, the non-LPA RNAi therapeutic is one of the following additional therapeutic agents: an HMG Co-A reductase inhibitor (statin), ezetimibe, a PCSK-9 inhibitor, a CTEP inhibitor, a therapy targeting ANGPTL3, a therapy targeting APOC3, and niacin, or a combination of any of the above.

[0249] In one embodiment, the iRNA agent is administered to the 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 described in the present disclosure as "non-diagnostic or non-therapeutic purposes" include, but are not limited to: as a positive control to screen other siRNA duplexes that inhibit the expression of LPA gene, or as an inhibition means to study the relationship between abnormal expression of LPA gene and diseases in the laboratory.

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

[0252] Table 1

[0253]

[0254] Examples

[0255] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0256] The experimental techniques and experimental methods used in the present embodiments are all conventional techniques and methods, and for example, the experimental methods not specifically described in the following examples are generally performed according to the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through commercial channels, unless otherwise specified.

[0257] Those skilled in the art can understand that, in various embodiments of the present disclosure, when the subject in the experiment is an siRNA conjugate, the inhibition rate, IC 50 , IC 40 of the experimental data and results can all correspondingly reflect the inhibition rate, IC 50 , IC 40 of the corresponding siRNA modifier of the siRNA conjugate. There is no obstacle to understanding for those skilled in the art.

[0258] Example 1: Inhibition of LPA gene by siRNA motif

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

[0260] 1.1 Synthesis of siRNA motif

[0261] Instrument and reagent: Genescript 192 P type DNA / RNA automatic synthesizer, its solid phase carrier is a general carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (cytiva manufacturer).

[0262] Preparation method:

[0263] According to the monomer concentration of 0.15 M, the following nucleotide monomer solutions were prepared with acetonitrile: 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] The nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction by solid-phase phosphoramidite method. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and hydroxyl protection. When the phosphate ester is used for the connection between two nucleotides, the four steps of deprotection, coupling, oxidation, and hydroxyl protection are included when the next nucleotide monomer is connected. When the phosphorothioate is used for the connection between two nucleotides, the four steps of deprotection, coupling, sulfurization, and hydroxyl protection are included when the next nucleotide monomer is connected.

[0267] The following steps are specifically used for preparation:

[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 is used as a deprotection reagent to remove the DMT protecting group, and then acetonitrile is used for cleaning.

[0271] (2) Coupling

[0272] 0.25 M 5-ethylthiotetrazole is used as an activator for the acetonitrile solution of each nucleotide monomer for coupling, and then acetonitrile is used for washing.

[0273] (3) Oxidation / sulfurization

[0274] Oxidation: 0.05 M iodine in pyridine / water (90 / 10) solution is used as an oxidizing agent for oxidation, and then acetonitrile is used for washing.

[0275] Sulfurization: 3% hydrogenated xanthate in pyridine solution is used as a sulfurizing agent for sulfurization, and then acetonitrile is used for washing.

[0276] (4) Hydroxyl protection

[0277] 10% acetic anhydride in tetrahydrofuran (CAP A) tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) is used as a hydroxyl protection reagent for hydroxyl protection, and then acetonitrile is used for washing.

[0278] The above operations are repeated, and the above steps are cycled according to the set nucleotide arrangement order to obtain the product of the sense strand or the product of the antisense strand with a specific sequence arrangement.

[0279] (5) 3% dichloroacetic acid in toluene is used as a deprotection reagent to remove the DMT protecting group of the last nucleotide, and then acetonitrile is used for cleaning.

[0280] (6) Aminolysis and purification

[0281] The reacted solid carrier was transferred to a reactor, concentrated ammonia (25-28%) was added, and the system was kept at 60°C for 12 h of aminolysis. After the system was cooled to room temperature, the mixture was filtered, the filter cake was eluted with a mixture of purified water and ethanol, the filtrates were combined, chromatographed, concentrated, and lyophilized to obtain the 2'-O-TBDMS-protected product.

[0282] (7) Removal of TBDMS

[0283] DMSO and triethylamine hydrofluoric acid were added to the obtained product, and the mixture was reacted at 60°C for 2 h. Then, an aqueous ammonium acetate solution was added to the reaction solution, the mixture was shaken and mixed, anhydrous ethanol was added, the mixture was shaken and mixed, and then the mixture was crystallized at -20°C for 8-12 h. After centrifugation, the supernatant was discarded, and the precipitate was eluted with anhydrous ethanol to obtain the unmodified single-stranded product.

[0284] (8) Annealing

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

[0286] 110 siRNA motifs in Table 2 were synthesized by the above method, in which APC is a positive control, which is the unmodified base sequence of the drug Olpasiran of Amgen, 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 for detecting the inhibitory effect of siRNA motifs on LPA gene

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

[0293] 1.2.1 Experimental materials

[0294] Table 3

[0295]

[0296] psiCHECK2-LPA plasmid construction: LPA gene mRNA full-length sequence (NM_007755.4) was cloned into dual luciferase plasmid psiCHECK TM -2 in the table, commissioned by Zunshou Biotech (Guangzhou) Co., Ltd., item number VB240227-1779ggq.

[0297] 1.2.2 Experimental method

[0298] 1) Plasmid transfection and cell plating

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

[0300] The psiCHECK2-LPA plasmid was diluted with Opti-MEM to 10 ng / μL. Huh7 cells were taken, washed with DPBS first, then trypsin was added for digestion, and the cell density was adjusted to 1×10 5 cells / mL. According to the ratio of Fugene-HD transfection reagent: 10 ng / μL of psiCHECK2-LPA plasmid dilution = 3:100 (volume ratio), mix well, incubate at room temperature for 10 min, then add to Huh7 cells, then seed in 96-well plates at a density of 10,000 cells per well, with 100 μL of culture medium per well. Huh7 cells were incubated in a 5% CO2, 37°C incubator overnight.

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

[0302] Day 1: siRNA treatment

[0303] Mix RNAiMAX transfection reagent and Opti-MEM at a ratio of 1.5:48.5 by volume to get mixture X, incubate at room temperature for 15 min, mix the siRNA solution with a starting concentration of 12 nM and 60 nM with the above mixture X at a volume ratio of 1:1 to get the corresponding mixture Y, incubate at room temperature for 15 min, then add 20 μL of the resulting mixture Y to 100 μL of fresh DMEM medium at a volume ratio of 1:5 and mix well to get the corresponding mixture Z, so that the final concentration of the siRNA to be tested is 1 nM and 5 nM, respectively. Discard the supernatant of the Huh7 cells cultured overnight in the 96-well plate in step 1), add 120 μL of the above mixture Z to each well of the 96-well plate, then place the 96-well plate in a CO2 cell incubator for 48 h.

[0304] 3) Cell sampling and fluorescence expression detection

[0305] Day 3: detection of reporter gene

[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 ratio calculated according to the above formula is denoted as a (experimental group), and the ratio of the control group is denoted as a (transfection reagent control group).

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

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

[0320] 2 transfection concentrations were set in 3 parallel detection holes, and the inhibition rate of each siRNA motif to LPA mRNA expression is shown in Table 4 below.

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

[0322]

[0323]

[0324] Example 2: Synthesis of alternately modified siRNA modifier

[0325] In order to improve the inhibition rate and stability, the siRNA motif in Table 2 is alternately modified with 2'-methoxy (2'-OMe) and 2'-fluoride (2'-F), and has 3', 5'-phosphorothioate bonds between the nucleotides at the 5' end and / or the 3' end.

[0326] 2.1 Synthesis of alternately modified siRNA modifier

[0327] The alternately modified rule in the present disclosure is that the nucleotides at the odd point positions of the sense strand and the nucleotides at the even point positions of the antisense strand are both modified with 2'-F, and the nucleotides at other point positions are modified with 2'-OMe. In addition, the alternately modified siRNA modifier in the present disclosure has 3', 5'-phosphorothioate bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 5' end of the sense strand; and has 3', 5'-phosphorothioate bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 5' end of the antisense strand, and has 3', 5'-phosphorothioate bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides at the 3' end of the antisense strand. The alternately modified siRNA modifier in the present disclosure is denoted as "-AL" after the numbering of the corresponding base sequence, as shown in Table 5.

[0328] Instrument and reagent: Genesee 192 P model DNA / RNA automatic synthesizer, its solid phase carrier is a general carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (cytiva manufacturer).

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

[0330] According to the monomer concentration of 0.15 M, the following nucleotide monomer solutions are prepared respectively using acetonitrile: DMT-A-OMe phosphoramidite monomer (formula 1), DMT-C-OMe phosphoramidite monomer (formula 2), DMT-G-OMe phosphoramidite monomer (formula 3) and DMT-U-OMe phosphoramidite monomer (formula 4), DMT-A-F phosphoramidite monomer (formula 5), DMT-C-F phosphoramidite monomer (formula 6), DMT-G-F phosphoramidite monomer (formula 7) and DMT-U-F phosphoramidite monomer (formula 8).

[0331]

[0332]

[0333]

[0334] By solid phase phosphoramidite method, the nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and hydroxyl protection. Among them, when the phosphate linkage is used between two nucleotides, when the next nucleotide monomer is connected, it includes four steps of deprotection, coupling, oxidation, and hydroxyl protection. When the phosphorothioate linkage is used between two nucleotides, when the next nucleotide monomer is connected, it includes four steps of deprotection, coupling, sulfurization, and hydroxyl protection.

[0335] (1) Deprotection

[0336] 3% dichloroacetic acid toluene solution is used as deprotection reagent to remove DMT protecting group, and then acetonitrile is used for cleaning.

[0337] (2) Coupling

[0338] 0.25 M 5-ethylthiotetrazole is used as an activator to couple the acetonitrile solution of each nucleotide monomer, and then acetonitrile is used for washing.

[0339] (3) Oxidation / Sulfurization

[0340] Oxidation: 0.05 M iodine in pyridine / water (90 / 10) solution is used as an oxidizing agent for oxidation, and then acetonitrile is used for washing.

[0341] Sulfurization: sulfurization was performed using a 3% hydrogen xanthate pyridine solution as the sulfurization agent, followed by washing with acetonitrile.

[0342] (4) Hydroxyl protection

[0343] Hydroxyl protection was performed using 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protection reagent, followed by washing with acetonitrile.

[0344] The above operations were repeated, and the above steps were performed in a set nucleotide arrangement sequence to obtain a specific sequence arrangement of the sense strand product or the antisense strand product.

[0345] (5) Deprotection of the last nucleotide DMT protecting group was performed using 3% dichloroacetic acid toluene solution as the deprotection reagent, followed by washing with acetonitrile.

[0346] (6) Amination and purification

[0347] The solid phase carrier was transferred to a reactor, concentrated ammonia water (25%-28%, mass percentage) was added, and amination was performed at 60°C for 12 h. The system was then cooled to room temperature, and the mixture was transferred to a filter tank for filtration. The filter cake was rinsed with a mixed solution of purified water and ethanol, and the filtrate was combined and passed through a chromatography column. After concentration, freeze-drying was performed to obtain the 2’-OMe and 2’-F modified single-stranded product.

[0348] (7) Annealing

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

[0350] The sense and antisense strands of the siRNAs listed in Table 5 were synthesized according to the above method, a total of 110 siRNAs. Among them, APC-AL is a positive control, which is the original sequence of the Amgen drug Olpasiran (its basic sequence is shown in Table 2) subjected to the above alternating modification, APC-OL is a positive control, which is the original sequence of the Amgen drug Olpasiran (its basic sequence is shown in Table 2) subjected to the original modification (the modification sequence of its sense strand is shown as SEQ ID NO: 219, and the basic sequence of its antisense strand is shown as SEQ ID NO: 220), ANC-AL is a negative control (its basic sequence is shown in Table 2), which is an alternatingly modified nonsense sequence.

[0351] APC-OL:

[0352] Sense (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 (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 represents: the nucleotide is DNA linked to the 20th nucleotide with a 3'-3' phosphodiester bond.

[0355] Example 3: Inhibition of LPA gene by alternately modified siRNA

[0356] The inhibition of LPA gene by the alternately modified siRNA synthesized in Example 2 was determined by two methods, dual-luciferase method and human primary hepatocyte transfection method.

[0357] 3.1 Detection by dual-luciferase method

[0358] The inhibition of LPA gene by the 110 siRNAs in Table 5 was determined by dual-luciferase method, and the experimental materials and methods were the same as those in Example 1, 1.2.1 and 1.2.2.

[0359] 3.1.1 Experimental results

[0360] The inhibition of LPA gene by the siRNAs in Table 5 was determined by the method described in Example 1, 1.2. The inhibition rates of the siRNAs at the concentrations of 1 nM and 5 nM are shown in Table 5 below. Among them, 41 siRNAs produced at least 50% inhibition of LPA gene expression at 5 nM. At the concentration of 1 nM, the inhibition rates of 21 siRNAs were higher than that of APC-AL, for example, the inhibition rate of A01501-AL was 47.9%, the inhibition rate of A02401-AL was 40.8%, the inhibition rate of A06601-AL was 38.5%, the inhibition rate of A04501-AL was 35.6%, and the inhibition rate of APC-AL was 35.2%.

[0361] Table 5 Inhibition rate (%) of alternately modified siRNA on LPA gene

[0362]

[0363]

[0364] 3.2 Inhibition of LPA gene by alternative modified siRNA modifiers detected by transfection of human primary hepatocytes

[0365] 19 of the 110 siRNA modifiers in Table 5 were selected (see Table 7) for their better activity. After the modifiers were transfected into human primary hepatocytes by liposome (Lipofectamine RNAiMAX), the inhibition rate of each sequence on LPA gene was detected by qPCR technology, and the IC 40 The siRNA transfection concentration was set to 20 nM as the starting point, 5-fold gradient dilution, and 6 concentrations (20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.006 nM) of siRNA working solution were prepared to detect the inhibition activity on LPA gene.

[0366] 3.2.1 Experimental materials

[0367] Table 6

[0368]

[0369] Solvent: sterilized RNase-free water.

[0370] 3.2.2 Experimental method

[0371] 1) Resuscitation of human primary hepatocytes

[0372] The culture medium was preheated at 37°C for more than 30 min. 120 μL of PBS was added to each well of the collagen-coated 96-well plate, and the PBS was aspirated after shaking several times. The cell freezing tube was taken out from the liquid nitrogen tank and placed in a 37°C water bath, and gently shaken until only a small amount of ice crystals remained in the tube. The cell suspension was poured into the resuscitation culture medium at one time. The inside wall of the freezing tube was rinsed with 1 mL of resuscitation culture medium for 2-3 times. The resuscitation culture medium cell suspension was inverted and mixed. Centrifugation at 150 g for 5 min at room temperature, and then the supernatant was discarded and the cells were resuspended with preheated plating medium. The cell counter was used for counting.

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

[0374] siRNA transfection complex preparation: Mix RNAiMAX transfection reagent and Opti-medium at a ratio of 1.5:23.5 by volume, and incubate at room temperature for 15 minutes to obtain solution A; dilute the siRNA modifier solution with Opti-MEM according to the transfection concentration, so that the prepared modifier concentration is 20 times the final transfection concentration, and the diluted solution is solution B; mix solution A and solution B at a ratio of 1:1 to obtain solution C, and incubate at room temperature for 15 minutes before adding the collagen-coated 96-well cell culture plate, and add 10 μL per well.

[0375] Cell dilution and plating: After the PHH cells are counted, the plating medium is added to the cell suspension according to the counting results, the cell concentration is adjusted to 5×10 5 ~6×10 5

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

[0377] 3) RNA extraction and reverse transcription

[0378] After transfection for 24 hours, the culture medium is removed and the cells are collected for RNA extraction. Total RNA is extracted using the Rneasy Mini Kit (QIAGEN-74106) according to the kit instructions. Then, cDNA is synthesized using the FastKing RT Kit (With gDNase) (TIANGEN-KR116-02) according to the instructions.

[0379] 4) RT-qPCR

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

[0381] 5) Data analysis

[0382] The ΔΔCt relative quantification method is used to calculate the RNA expression level of the target gene in the sample according to the Ct value of each sample. The relative expression of the target gene is represented by 2 -ΔΔCt .

[0383] The calculation formula is as follows:

[0384] ​ΔCT = average Ct value of target gene - average Ct value of internal control gene;

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

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

[0387] Inhibition rate = (1 - sample relative expression / average expression of RNAiMAX control) x 100%

[0388] The dose-effect curve was fitted by using GraphPad Prism software (Nonlinear four parameter logistic equations).

[0389] 3.2.3 Experimental results

[0390] After 3 qPCR detections, the inhibition rates of each siRNA modifier on LPA gene of human primary hepatocytes under different concentrations were shown in Table 7. Among them, A04101-AL, A06601-AL, A04501-AL, A01501-AL, A02401-AL and A04401-AL had 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 40 of the above 6 siRNA modifiers was 0.036 nM, which was better than that of APC-AL, for example, the IC 40 of B04101-AL was 7.461 nM.

[0391] Table 7 Inhibition rate of alternative modifier on LPA of human primary hepatocytes under different concentrations and IC 40

[0392]

[0393] Example 4: Comparison of inhibition rates of siRNA motifs in the present disclosure and similar sequences in the prior art

[0394] In this example, the inhibition rates of 6 siRNA motifs A01501, A02401, A04101, A04401, A04501 and A06601 in the present disclosure on LPA gene were compared with similar sequences in the prior art.

[0395] 1. Experimental materials

[0396] Test samples: The siRNA motifs in Table 8 were synthesized using the synthetic method of 1.1 in Example 1.

[0397] Table 8

[0398]

[0399] 2. Experimental methods and results

[0400] Experimental materials and experimental methods refer to 1.2.1 and 1.2.2 in Example 1, and the siRNA transfection concentration is 5 nM.

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

[0402] Table 9

[0403]

[0404] From the above table, it can be seen that the inhibition rates of the six basic sequences (i.e. A01501, A02401, A04101, A04401, A04501, A06601) of the present disclosure and similar motifs of the prior art are compared. The results show that under the same concentration conditions, the inhibition rates of the above six basic sequences of the present disclosure on LPA mRNA expression in Huh7 cells are all better than those of similar sequences of the prior art. For example, compared with the similar sequence A04101P2 of the prior art, the inhibition rate of A04101 of the present disclosure on LPA gene is increased by 76.3%; compared with the similar sequence A01501P, the inhibition rate of A01501 of the present disclosure on LPA gene is increased by 42.6%.

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 is a sequence as shown in SEQ ID NO: 124 or a modified sequence thereof; and the sense strand is a sequence as shown in SEQ ID NO: 15 or a modified sequence thereof; The odd-numbered positions of the modified sequence of the antisense strand are all 2'-methoxy modified, and the even-numbered positions are all 2'-fluoro modified; the modified sequence of the antisense strand 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; The odd-numbered positions of the modified sequence of the sense chain are all 2'-fluoro modified, and the even-numbered positions are all 2'-methoxy modified, and the first and second nucleotides and the second and third nucleotides at the 5' end of the modified sequence of the sense chain are connected by 3',5'-phosphorothioate bonds.

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

3. A siRNA conjugate, characterized in that The conjugate comprises the siRNA duplex according to claim 1 or 2, and a conjugated group connected thereto.

4. A nucleic acid-protein complex, characterized in that The nucleic acid-protein complex comprises the double-stranded region of the siRNA duplex according to claim 1 or 2, and a nuclease; or, the nucleic acid-protein complex comprises the antisense strand of the siRNA duplex according to claim 1 or 2, and a nuclease.

5. The nucleic acid-protein complex according to claim 4, wherein The nuclease is AGO protein.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the siRNA duplex according to claim 1 or 2, the conjugate according to claim 3, or the nucleic acid-protein complex according to claim 4 or 5, and a pharmaceutically acceptable carrier.

7. Use of the siRNA duplex according to claim 1 or 2, the conjugate according to claim 3, the nucleic acid-protein complex according to claim 4 or 5, or the pharmaceutical composition according to claim 6 in the preparation of a reagent and a kit for inhibiting LPA gene expression.

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