Rna i agents targeting pcsk9 and uses

Abstract

The application belongs to the field of biological medicine, and particularly relates to an RNAi reagent targeting PCSK9 and use. The application significantly improves the inhibition effect of the RNAi reagent on PCSK9 by improving a modification mode, and thus has better application potential in preventing or treating diseases related to PCSK9.

Description

Cross-references to related applications

[0001] This invention claims priority to Chinese Patent Application No. 2024116590271, filed on November 19, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biomedicine, specifically relating to RNAi reagents targeting PCSK9 and their uses. Background Technology

[0003] Proprotein Convertase Subtilisin / Kexin type 9 (PCSK9) is a protein-converting enzyme that plays a crucial role in the liver. It regulates LDL cholesterol levels in the blood by binding to and promoting the degradation of low-density lipoprotein (LDL) receptors. Overexpression of PCSK9 leads to a reduction in LDL receptors, resulting in elevated LDL cholesterol levels in the blood and increasing the risk of cardiovascular diseases such as hypercholesterolemia, atherosclerosis, coronary heart disease, and stroke. Therefore, PCSK9 inhibitors have become important drug targets for the treatment and prevention of these diseases.

[0004] PCSK9 inhibitors affect the function and quantity of PCSK9 in the body through different pathways, including blocking the binding of PCSK9 to LDLR, interfering with PCSK9 secretion, and inhibiting PCSK9 expression. Currently, four PCSK9 inhibitory therapies (PCSK9-iT) have been approved for clinical use: monoclonal antibodies (Evolocumab, Alirocumab, and Tafolecimab) and siRNA (Inclisiran). These treatments can significantly reduce LDL-C levels, lowering them by approximately 50-60% compared to statin therapy alone. Among these, RNAi drugs (siRNA) have the advantage of a longer duration of action compared to antibodies. Therefore, the research and development of siRNAs targeting PCSK9 is of great significance. However, there is still room for further improvement in the efficacy of current siRNAs in inhibiting PCSK9. Summary of the Invention

[0005] The first objective of this invention is to provide an RNAi reagent comprising a sense strand and an antisense strand; wherein the antisense strand contains the nucleotide sequence from position 1 to 21 of 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID No. 1); the sense strand contains a nucleotide sequence at least partially complementary to the antisense strand; all nucleotides in the antisense strand are modified nucleotides and satisfy at least one of the following conditions I) to II): I) the antisense strand does not contain three consecutive identical chemical modifications at positions 1 to 20 of the sequence shown in SEQ ID No. 1; II) the antisense strand is at position 2' of the first nucleotide of the sequence shown in SEQ ID No. 1. Fluoronucleotides.

[0006] The present invention also provides a cell containing the aforementioned RNAi reagent.

[0007] The present invention also provides a pharmaceutical composition comprising the aforementioned RNAi reagent.

[0008] A second objective of the present invention is to provide a method for inhibiting the expression of PCSK9 in cells, the method comprising: contacting the cells with the RNAi reagent or the pharmaceutical composition thereof to inhibit the expression of PCSK9 in the cells.

[0009] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the treatment and / or prevention of diseases associated with PCSK9.

[0010] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the preparation of medicaments for the treatment and / or prevention of diseases associated with PCSK9.

[0011] This invention significantly enhances the inhibitory effect of RNAi reagents on PCSK9 by improving the modification method, thus showing greater application potential in the prevention or treatment of PCSK9-related diseases. Detailed Implementation

[0012] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the invention. Those skilled in the art can make various modifications and variations to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment can be used in another embodiment to produce further embodiments.

[0013] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0014] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0015] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0016] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0017] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% can fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0018] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0019] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0020] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" are merely descriptions of more effective implementation methods or embodiments, and should be understood not to limit the scope of protection of this invention.

[0021] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, then each "optional" or "optional" term is independent.

[0022] In this invention, the term "RNAi reagent" (also known as "RNAi trigger") means a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting (e.g., under appropriate conditions, degrading or inhibiting) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. As used herein, RNAi reagents may function via RNA interference mechanisms (i.e., by interaction with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC) or via any alternative mechanism or pathway. Although it is considered that RNAi reagents function primarily through RNA interference mechanisms as used herein, the disclosed RNAi reagents are not bound to or limited to any particular pathway or mechanism of action. The RNAi reagents disclosed herein consist of a sense strand and an antisense strand, and include, but are not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi reagent described herein is at least partially complementary to the target mRNA (i.e., PCSK9 mRNA). The RNAi reagent may include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0023] In this invention, when referring to the expression of a given gene, the terms “silence,” “reduction,” “inhibition,” “downregulation,” or “knockdown” mean, as measured by the level of RNA transcribed from the gene or the level of polypeptides, proteins, or protein subunits translated from mRNA in a cell, cell population, tissue, organ, or subject in which the gene is transcribed, when the cell, cell population, tissue, organ, or subject is treated with the RNAi reagent described herein, the expression of the gene is reduced compared to a second cell, cell population, tissue, organ, or subject who has not been treated in this way.

[0024] In this invention, "completely complementary" means that in a hybridization pair of nucleobase or nucleotide sequences, all (100%) bases in the adjacent sequence of the first oligonucleotide hybridize with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0025] In this invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all of the bases in the adjacent sequence of the first oligonucleotide hybridizes with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0026] In this invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all of the bases in the adjacent sequence of the first oligonucleotide hybridizes with the same number of bases in the adjacent sequence of the second oligonucleotide. The adjacent sequence may comprise all or part of the first or second nucleotide sequence.

[0027] In this invention, the term "at least partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, the first oligonucleotide and the second oligonucleotide are partially complementary, substantially complementary, or completely complementary.

[0028] In this invention, the term "treatment" refers to a method or procedure taken to provide relief or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, preventative treatment, and / or suppression or reduction of the number, severity, and / or frequency of one or more disease symptoms in a subject.

[0029] In this invention, the term "link" means the combination of two compounds or molecules through a covalent bond. Unless otherwise stated, as used herein, the term "link" may refer to a link between a first compound and a second compound, with or without any intermediate atoms or groups of atoms.

[0030] In this invention, the term "the ...th nucleotide position of the sequence shown in SEQ ID No. ..." refers to the position relative to the specific sequence. When the position of a nucleotide in the sequence changes (e.g., after adding a nucleotide at the 5' end), those skilled in the art can still identify the position of the corresponding nucleotide in the specific sequence according to the nucleotide corresponding to the position of the specific sequence in this invention, which also falls within the protection scope of this invention.

[0031] In this invention, the letters 'G', 'C', 'A', and 'U' generally represent nucleotides containing guanine, cytosine, adenine, or uracil bases, respectively. 'T' and 'dT' are used interchangeably, specifically referring to deoxyribonucleotides with thymine (such as deoxyribothymine, 2'-deoxythymidine, or thymidine) as a base. It should be noted that the terms 'ribonucleotide', 'nucleotide', or 'deoxyribonucleotide' also encompass modified nucleotides (see subsequent description) or their alternative substitution groups. Those skilled in the art will know that when guanine, cytosine, adenine, or uracil is replaced with other groups, the overall function of the oligonucleotide remains largely unaffected as long as the substitution group in the nucleotide maintains its base-pairing ability. For example (but not limited to), nucleotides containing inosine bases (I) can pair with nucleotides containing adenine, cytosine, or uracil. Therefore, the uracil, guanine, or adenine nucleotides in the nucleotide sequence of this invention can be replaced with nucleotides containing substitution groups such as inosine. Sequences with such substitutional bases are all within the scope of protection of this invention.

[0032] In this invention, the "modified nucleotide" is selected from 2' O Methylnucleotide, 2' Fluoronucleotides, 2' Deoxynucleotides, 2', 3' Open-ring nucleotide mimics, locked nucleotides, 2' F Arabinonucleotides, 2' Methoxyethyl nucleotide, debased nucleotide, ribitol, reverse nucleotide, reverse 2' O Methylnucleotide, reverse 2' Deoxynucleotides, 2' Amino-modified nucleotides, 2' Alkyl-modified nucleotides, morpholine nucleotides, peptide nucleic acids, glycerol nucleic acids, triazine-cyclic DNA, nucleotides containing non-natural bases, nucleotides containing vinyl phosphonates, nucleotides containing cyclopropyl phosphonates, and 3'-nucleotides. O One or more of methyl nucleotides. In some specific embodiments, the modified nucleotide is selected from 2'... O Methylnucleotide, 2' Fluoronucleotides, 2' Deoxynucleotides.

[0033] In this invention, the "chemical modification" includes, but is not limited to, the following modifications: 2'-O-methyl modification, 2'-fluorine modification, 2'-deoxy modification, 2'-F-arabinose modification, 2'-methoxyethyl modification, 2'-amino modification, 2'-alkyl modification; 3'-O-methyl modification; locked nucleic acid methylene bridging modification; debasing modification; non-natural base modification; vinyl phosphonate modification; cyclopropyl phosphonate modification. In some specific embodiments, the chemical modification is selected from 2'-O-methyl modification and 2'-fluorine modification.

[0034] RNAi reagents The first objective of this invention is to provide an RNAi reagent comprising a sense strand and an antisense strand; wherein the antisense strand contains the nucleotide sequence from position 1 to 21 of 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID No. 1); the sense strand contains a nucleotide sequence at least partially complementary to the antisense strand (e.g., partially complementary, substantially complementary, or fully complementary); all nucleotides in the antisense strand are modified nucleotides and satisfy at least one of the following conditions I) to II): I) the antisense strand does not contain three consecutive identical chemical modifications at positions 1 to 20 of the sequence shown in SEQ ID No. 1; II) the antisense strand is at position 2' of the first nucleotide of the sequence shown in SEQ ID No. 1. Fluoronucleotides. This invention has found that by modifying the antisense strand in the manner described above, the inhibitory effect of RNAi reagents on PCSK9 can be significantly improved.

[0035] In some implementations, the antisense chain satisfies the condition described in I) above.

[0036] In some implementations, the antisense chain satisfies the condition described in II) above.

[0037] In some implementations, the antisense chain satisfies conditions I) and II) above.

[0038] In some embodiments, the nucleotides modified at other positions in the antisense strand are each independently selected from 2' O Methylnucleotide, 2' Fluoronucleotides.

[0039] In some embodiments, the antisense strand contains the nucleotide sequence of 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID No. 1).

[0040] In some embodiments, the antisense strand is 2' at positions 4 and 5 of the sequence shown in SEQ ID No. 1. Fluoronucleotides, with the 6th nucleotide at the 2' position. O Methylnucleotide. Compared to these three positions, all are 2'. In the case of fluoronucleotides, this modification method has a significantly better inhibitory effect on PCSK9, and the advantage in in vivo is even more prominent.

[0041] In some embodiments, the antisense strand is positioned at 2' of nucleotides 2 and 14 of the sequence shown in SEQ ID No. 1. Fluoronucleotides.

[0042] In some embodiments, the antisense strand is 2' at nucleotide positions 2, 8, 10, 12, 14, 16, and 18 of the sequence shown in SEQ ID No. 1. Fluoronucleotides.

[0043] In some embodiments, the positive strand contains the nucleotide sequence 5'-CUAGACCUGUUUUGCUUUUGU-3' (SEQ ID No. 2) or 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID No. 3).

[0044] In some preferred embodiments, all nucleotides in the positive strand are modified nucleotides, and the positive strand has a 2' position at the first and / or third nucleotide position as shown in SEQ ID No. 2 or 3. Fluoronucleotide; the positive strand is at position 2' of the 11th nucleotide in the sequence shown in SEQ ID No. 3. Deoxythymidine. The above modifications to the sense strand can further improve the inhibitory effect of RNAi reagents on PCSK9.

[0045] In some specific embodiments, all nucleotides in the positive strand are modified nucleotides, and the positive strand has a 2' position at the first nucleotide of the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides.

[0046] In some specific embodiments, all nucleotides in the positive strand are modified nucleotides, and the positive strand is at position 2' of the third nucleotide in the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides.

[0047] In some preferred embodiments, all nucleotides in the positive strand are modified nucleotides, and the positive strand has 2' positions at the first and third nucleotide positions as shown in SEQ ID No. 2 or 3. Fluoronucleotides.

[0048] In some embodiments, the nucleotides modified at other positions in the positive strand are each independently selected from 2' O Methylnucleotide, 2' Fluoronucleotides.

[0049] In some embodiments, the positive strand contains the nucleotide sequence 5'-CUAGACCUGUUUUGCUUUUGU-3' (SEQ ID No. 2), and the positive strand is 2' at the 11th nucleotide position of the sequence shown in SEQ ID No. 2. Fluoronucleotides. This invention also discovered that when the 11th nucleotide of the sequence shown in SEQ ID No. 2 is at position 2'... When fluoronucleotides are used, the position is 2' relative to this position. The presence of deoxythymidine (as shown in SEQ ID No. 3) can further significantly improve the inhibitory effect of RNAi reagents on PCSK9.

[0050] In some embodiments, the positive strand is positioned at the 10th nucleotide of the sequence shown in SEQ ID No. 2 or 3 at a 2' position. Fluoronucleotides. This invention has found that the above-mentioned modification methods can also further improve the inhibitory effect of RNAi reagents on PCSK9.

[0051] In a preferred embodiment, the positive strand contains the nucleotide sequence 5'-CUAGACCUGUUUUGCUUUUGU-3' (SEQ ID No. 2), and the positive strand is 2' at positions 10 and 11 of the sequence shown in SEQ ID No. 2. Fluoronucleotides. The corresponding positions are 2'. O methyl nucleotides and 2' In the case of fluoronucleotides, this sequence and modification method have a significantly better inhibitory effect on PCSK9.

[0052] In some embodiments, the positive strand is 2' at the 7th and / or 9th nucleotide position of the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides.

[0053] In some embodiments, the positive strand is independently selected from 2' at positions 8 and 14 of the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides, 2' O Methyl nucleotides, and nucleotides modified at other positions are 2'. O Methyl nucleotide.

[0054] In some embodiments, the last three nucleotides at the 5' end of the positive strand contain at least one (e.g., one or two) phosphate thioester nucleotide inter-bonds.

[0055] In some specific embodiments, the last three nucleotides at the 5' end of the positive strand contain two phosphate-thioester nucleotide bonds. In some specific embodiments, the last two nucleotides at the 5' end of the positive strand contain one phosphate-thioester nucleotide bond. In some specific embodiments, the third-to-last and second-to-last nucleotides at the 5' end of the positive strand contain one phosphate-thioester nucleotide bond.

[0056] In some embodiments, the last three nucleotides at the 5' end of the antisense strand contain at least one (e.g., one or two) phosphate thioester nucleotide bonds, and the last five nucleotides at the 3' end of the antisense strand contain at least one (e.g., one, two, three, or four) phosphate thioester nucleotide bonds.

[0057] In some specific embodiments, the last three nucleotides at the 5' end of the antisense strand contain two phosphate-thioester nucleotide bonds. In some specific embodiments, the last two nucleotides at the 5' end of the antisense strand contain one phosphate-thioester nucleotide bond. In some specific embodiments, the third-to-last and second-to-last nucleotides at the 5' end of the antisense strand contain one phosphate-thioester nucleotide bond.

[0058] In some specific embodiments, the last three nucleotides at the 3' end of the antisense strand contain two phosphate-thioester nucleotide bonds. In some specific embodiments, the fourth to second-to-last nucleotides from the 3' end of the antisense strand contain two phosphate-thioester nucleotide bonds. In some specific embodiments, the fifth to third-to-last nucleotides from the 3' end of the antisense strand contain two phosphate-thioester nucleotide bonds. In some specific embodiments, the last two nucleotides at the 3' end of the antisense strand contain one phosphate-thioester nucleotide bond. In some specific embodiments, the third and second-to-last nucleotides from the 3' end of the antisense strand contain one phosphate-thioester nucleotide bond. In some specific embodiments, the fourth to third-to-last nucleotides from the 3' end of the antisense strand contain one phosphate-thioester nucleotide bond. In some specific embodiments, the antisense strand contains a phosphate thioester nucleotide bond between the fifth to fourth nucleotides from the end of its 3' end.

[0059] In some embodiments, the 5' end and / or 3' end of the positive chain contains a reversed abasic linker (or inverted abasic linker).

[0060] In some embodiments, the 3' end of the antisense chain contains at least one (e.g., one, two, or three) 2' ends. Deoxythymidine.

[0061] In some embodiments, the 3' end of the antisense chain contains at least one (e.g., one, two, or three) 2' ends. O Methyluridine.

[0062] Those skilled in the art can combine the above-described embodiments with common sense to obtain more embodiments of the RNAi reagent of the present invention.

[0063] In some specific implementations, the antisense and sense strands mentioned above can be combined to obtain RNAi reagents (siRNA) with sense and antisense strand sequences as shown in Table 1.

[0064] Table 1

[0065] In some preferred embodiments, the antisense strand contains nucleotides 1 to 21 of 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13). When the antisense strand has the above sequence, the RNAi reagent exhibits better inhibitory effects against PCSK9.

[0066] In some preferred embodiments, the sense strand contains the nucleotide sequence 5'-CfsusAfgacCfuGfUfUfuugcuuuugu-3' (SEQ ID No. 4), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13).

[0067] In some preferred embodiments, the sense strand contains the nucleotide sequence 5'-CfsusAfgacCfuGfUfUfuuGfcuuuugu-3' (SEQ ID No. 5), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13).

[0068] In some preferred embodiments, the sense strand contains the nucleotide sequence 5'-CfsusAfgacCfUfGfUfUfuuGfcuuuugu-3' (SEQ ID No. 6), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13).

[0069] In some preferred embodiments, the sense strand contains the nucleotide sequence 5'-CfsusAfgacCfuGfudTuugcuuuugu-3' (SEQ ID No. 7), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13).

[0070] In the sequences of this invention, nucleotides represented by lowercase letters indicate that the nucleotide is 2'. O Methyl nucleotide; f indicates that the nucleotide adjacent to it on the left is 2'. Fluoronucleotides; dT indicates that the nucleotide is 2' Deoxythymidine; s represents the connection between two adjacent nucleotides via a phosphothioester bond; Ral represents a reversed base deletion linker.

[0071] In some embodiments, when the sequence identity of the sense or antisense strand in the RNAi reagent is less than 100% or differs by more than one nucleotide from the corresponding sequence mentioned in this invention, it still has an inhibitory effect on PCSK9 that is similar to (e.g., still has an efficacy equivalent to 80-120%, 85-115%, or 90-110% of the corresponding sequence) or equivalent to (e.g., still has an efficacy equivalent to 95-105% of the corresponding sequence). For example, the two bases at the 3' end of the antisense strand (such as the sequence shown in SEQ ID NO: 1) are replaced with UU, CC, GG, UG, or any combination of two nucleic acids; or, for example, a 2' modification is performed at a modification site not specifically mentioned in this invention. O Methylnucleotides and 2' The substitution between fluoronucleotides. Such nucleic acid sequences are also within the scope of protection of this invention.

[0072] The RNAi reagent according to the present invention can be obtained by conventional methods in the art, such as solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis is available through commercially available custom services and is therefore commercially available. The modified nucleotide groups can be introduced using nucleotide monomers with corresponding modifications.

[0073] Based on the RNAi reagent (siRNA) synthesized above, the present invention can further construct shRNA expression plasmids with the same or similar functions as the above RNAi reagents. The method for constructing the expression plasmid is well known to those skilled in the art and will not be described in detail here.

[0074] In some embodiments, the RNAi reagent further comprises at least one ligand.

[0075] Based on common knowledge in the art, the sense and antisense strands of this invention exhibit superior inhibitory effects when applied to different targeted drug delivery systems. In other words, the efficacy advantage of the modified sequences in this invention does not depend on the choice of target vector. To further improve the bioavailability and therapeutic effect of siRNA, this invention also optimizes the targeted drug delivery system, resulting in the following technical solution.

[0076] In some embodiments, the at least one ligand is attached to one or more nucleotides of the antisense or sense strand, the nucleotides being selected from 5' terminal nucleotides, 3' terminal nucleotides, and / or any nucleotide within the strand.

[0077] In some specific embodiments, the at least one ligand is attached to the 3' terminal nucleotide of the positive strand.

[0078] In some embodiments, the ligand is a GalNAc derivative.

[0079] In some embodiments, the ligand is one or more GalNAc derivatives linked by a single-chain, double-chain, or triple-chain branched ligand.

[0080] In some embodiments, the RNAi reagent comprises a compound with the structure shown in Formula I:

[0081] Formula I In the formula, Nu represents a nucleic acid composed of the sense strand and the antisense strand. This targeted drug delivery system utilizes the structural characteristics on its left side to improve the cell penetration ability of nucleic acid drugs (Nu), enhance their stability within cells, and has a simple preparation process and strong practicality.

[0082] Cell and drug compositions The present invention also provides a cell containing the aforementioned RNAi reagent.

[0083] The cells can be used for gene function research, disease model research, or drug screening.

[0084] In some embodiments, the cells do not develop into an animal individual. In some specific embodiments, the cells may be microbial cells or animal cells, but the animal cells are not animal embryonic stem cells or cells at various stages of formation and development (e.g., germ cells, fertilized egg cells, etc.).

[0085] The present invention also provides a pharmaceutical composition comprising the aforementioned RNAi reagent.

[0086] The pharmaceutical composition can be prepared using conventional methods from the RNAi reagent and the pharmaceutically acceptable carrier. For example, the pharmaceutical composition can be an injection solution. The injection solution can be used for subcutaneous, intramuscular, or intravenous injection.

[0087] According to the pharmaceutical composition of the present invention, there are no particular requirements for the amount of RNAi reagent and pharmaceutically acceptable carrier. Generally, the content of the pharmaceutically acceptable carrier can be 1-100,000 parts by weight relative to 1 part by weight of the RNAi reagent (e.g., 1 part by weight, 5 parts by weight, 10 parts by weight, 50 parts by weight, 100 parts by weight, 500 parts by weight, 1000 parts by weight, 5000 parts by weight, 10000 parts by weight, 50000 parts by weight, 100000 parts by weight, or any value between any two of the above).

[0088] According to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier can be any of the carriers conventionally used in the art, for example, it can include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator. The pH buffer can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, preferably a phosphate buffer with a pH of 5.5-8.5. The protective agent can be at least one of inositol, sorbitol, and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protective agent can be 0.01-30% by weight (e.g., 0.01% by weight, 0.05% by weight, 0.1% by weight, 0.5% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, or any value between any two of the above). The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliohms / kg. The content of the osmotic pressure regulator can be determined by those skilled in the art based on the desired osmotic pressure.

[0089] According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any type of liposome capable of encapsulating nucleic acids, and its diameter can be 25-1000 nm, including but not limited to cholesterol and its analogues or derivatives.

[0090] The dosage of the pharmaceutical composition described in this invention can be a conventional dosage in the art, which can be determined based on various parameters, particularly the age, weight, and sex of the subject. For example, for female mice aged 3-4 months and weighing 25-30g, the dosage of the pharmaceutical composition can be 0.01-100 mg / kg body weight, preferably 1-10 mg / kg body weight, based on the amount of the RNAi reagent in the pharmaceutical composition.

[0091] Methods and uses The present invention also provides a method for inhibiting the expression of PCSK9 in cells, the method comprising: contacting the cells with the RNAi reagent or the pharmaceutical composition to inhibit the expression of PCSK9 in the cells.

[0092] In some implementations, the cells are in a subject, such as a human subject, a subject with PCSK9-related disease, or a subject who needs to prevent the risk of PCSK9-related disease.

[0093] In some embodiments, the cells are located in vitro. The method is based on research purposes or is used to construct animal models.

[0094] In some embodiments, contacting the cells with the nucleic acid inhibits PCSK9 expression by at least 50%, 60%, 70%, 80%, 90%, or 95% (e.g., compared to PCSK9 expression levels prior to the first contact of the cells with the nucleic acid; e.g., before administering a first dose of the nucleic acid to the subject). In some embodiments, inhibiting PCSK9 expression reduces PCSK9 protein levels in a subject's serum sample by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., compared to PCSK9 expression levels prior to the first contact of the cells with the nucleic acid.

[0095] The present invention also provides the use of the described RNAi reagent or the described pharmaceutical composition in the treatment and / or prevention of PCSK9-related diseases. Specifically, a method for treating and / or preventing PCSK9-related diseases includes administering the described RNAi reagent or the described pharmaceutical composition to a subject.

[0096] The present invention also provides the use of the RNAi reagent or the pharmaceutical composition described herein in the preparation of medicaments for the treatment and / or prevention of diseases associated with PCSK9.

[0097] In some implementations, the disease is: (i) a disease associated with enhanced or elevated PCSK9; or (ii) a disease that would benefit from reduced PCSK9 expression.

[0098] In some embodiments, the disease is a cardiovascular disease. Preferably, the cardiovascular disease is selected from one or more of hyperlipidemia, hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, and atherosclerosis.

[0099] In this invention, the subject can be a mammal, including primates (such as humans, non-human primates such as monkeys and chimpanzees), non-primates (such as cattle, pigs, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, or mice), or birds. In some embodiments, the subject is preferably a primate, and more preferably a human.

[0100] It can be administered via various routes, depending on whether local or systemic treatment is required. Dosage can be referred to above and will not be repeated here.

[0101] In some embodiments, administration can be local (e.g., percutaneous patch), pulmonary, such as by inhalation or blowing of powder or spray, including via nebulizer; intratracheal, nasal, epidermal, and percutaneous, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, such as via a transplant device; or intracranial, such as via intraparenchymal, intrathecal or intracardiac administration.

[0102] In some embodiments, the RNAi reagent or the pharmaceutical composition is administered to the subject via subcutaneous, intravenous, and / or intramuscular administration.

[0103] Example The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0104] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0105] Example 1 The siRNAs listed in Table 2 were obtained after preliminary screening. To further determine the activity of the siRNAs, a dose-response experiment was conducted on the siRNAs in primary monkey hepatocytes. Specifically, the siRNA samples were dissolved in 100 μL of enzyme-free sterile water to prepare a 100 μM solution. 30 μL of the 100 μM test solution was added to 70 μL of PMonH plating medium to dilute it to a 30 μM solution as the working solution for the 30 nM final concentration group. The 30 μM test solution was then 3-fold diluted at 8 concentration points using PMonH plating medium to obtain the final working solution concentrations of 4.6 nM, 13.7 nM, 41.2 nM, 123.4 nM, 370.4 nM, 1111.1 nM, 3333.3 nM, and 10000 nM. Primary monkey hepatocytes were removed from liquid nitrogen, thawed and revived at 37°C, washed with serum-containing PMonH plating medium, counted, and centrifuged. After removing the supernatant, the cells were diluted to 300 kJ / mL with fresh serum-containing PMonH plating medium. 90 μL of the diluted cell solution was then plated onto 96-well cell culture plates, resulting in 30 kJ cells per well. The prepared sample working solution was added to the cell solution to achieve final concentrations of 0.46 nM, 1.37 nM, 4.12 nM, 12.34 nM, 37.04 nM, 111.11 nM, 333.33 nM, and 1000 nM. The plates were then incubated at 37°C for 48 hours in a 5% CO2 incubator. Forty-eight hours later, all culture medium was aspirated from the 96-well plate, washed with 1×PBS buffer, and 50 μL of prepared Cells to CT lysis buffer (as recommended by the manufacturer) was added and mixed. After standing for 10 min, 2.5 μL of stop solution was added to terminate the reaction for 2 min. RT-PCR was performed according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, catalog number: 4368814), with each reaction containing 10 μL of lysed liquid. Gene expression was quantified using real-time fluorescence PCR. The TaqMan probe for monkey PCSK9 was Mf03418189_m1, and the probe for the internal control gene (monkey PPIB) was Mf02802985_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 95℃ for 20 seconds per cycle, followed by 40 cycles of 95℃ for 1 second and 60℃ for 20 seconds. The real-time PCR instrument used was a QuantStudio™ 6 Pro real-time quantitative PCR system (Thermo Fisher). PCSK9 gene expression was calculated as 2^-ΔΔCt, and monkey PPIB gene expression was used as an internal control.The expression level of the PCSK9 gene was calculated as a percentage of the cell group with culture medium alone, and the IC50 value was calculated. The results are shown in Table 3.

[0106] Table 2

[0107] Table 3

[0108] The results show that SNK-6809 has a significantly better inhibitory effect on the PCSK9 gene. Further optimization of its modification method was conducted, and the modified sequences shown in Table 4 were obtained. To confirm its inhibitory effect, 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 10... was added to a 96-well cell culture dish. 4Hep3B (Procell, Cat# CL-0102) cells were cultured overnight at 37°C in a 5% CO2 cell culture vessel. RNAiMAX (1.5 μL / well) and small interfering nucleic acids from Table 4 were added to Opti-MEM medium to bring the final concentration per well to 1 nM or 10 nM. Cells were then cultured at 37°C in 5% CO2 for 48 hours. To extract RNA, the cell culture supernatant was aspirated, washed with PBS, and then 50 μL of prepared lysis buffer (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)) was added and mixed. After standing for 10 min, 2.5 μL of stopsolution was added to terminate the reaction for 2 min. RT-PCR was performed according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (ThermoFisher, Catalog No.: 4368814), with each reaction containing 10 μL of lysed liquid. Gene expression was quantified using real-time fluorescence PCR. The human PCSK9 TaqMan probe was Hs00545399_m1, and the probe for the internal control gene (human HPRT1) was Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 95℃ for 20 seconds per cycle, followed by 40 cycles of 95℃ for 1 second and 60℃ for 20 seconds. The real-time fluorescence PCR system used was a QuantStudio™ 6 Pro real-time fluorescence quantitative PCR system (Thermo Fisher). PCSK9 gene expression was calculated as 2^-ΔΔCt, with human HPRT1 gene expression used as an internal control. PCSK9 gene expression levels were expressed as a percentage relative to the control group (RNAiMAX only). The results are shown in Table 5.

[0109] Table 4

[0110] Table 5

[0111] Example 2 To further explore optimal modification methods, a free uptake experiment was conducted in human primary hepatocytes with the hepatocyte-targeting compound Tri-GalNAc (the structure of which is shown in Formula I, and the relevant sequences are shown in Table 6). siRNA-GalNAc samples were dissolved in 100 μL of enzyme-free sterile water to prepare a 10000 μM solution, which was then diluted to 1000 and 100 μM concentrations using inVitroGRO Plating Medium. Human primary hepatocytes were removed from liquid nitrogen, thawed and revived at 37°C, washed and counted with serum-containing inVitroGRO Plating Medium, centrifuged, and after removing the supernatant, diluted to 350 kJ / mL with fresh serum-containing inVitroGRO Plating Medium. 90 μL of the diluted cell solution was then plated onto 96-well cell culture plates, resulting in 35 kJ cells per well. The prepared siRNA working solution was added to the cell solution to achieve final concentrations of 100 and 1000 nM. The plates were then incubated at 37°C for 48 hours in a 5% CO2 incubator. After 48 hours, all culture medium was aspirated from the 96-well plates, and the plates were washed with 1×PBS buffer. RNA was extracted according to the RNeasy Mini Kit (QIAGEN) protocol. RT-PCR was performed according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (ThermoFisher, catalog number: 4368814), with each reaction containing 10 μL of lysed liquid. Gene expression was quantified using real-time fluorescence PCR. The TaqMan probe for human PCSK9 was Hs00545399_m1, and the probe for the internal control gene (human HPRT) was Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 95°C for 20 seconds per cycle, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. The real-time PCR instrument used was a QuantStudio™ 6 Pro real-time quantitative PCR system (Thermo Fisher). PCSK9 gene expression was calculated as 2^-ΔΔCt, with HPRT gene expression used as an internal control. PCSK9 gene silencing levels were calculated as a percentage of the control group (cells with only culture medium). The remaining PCSK9 mRNA expression values ​​are shown in Table 7.

[0112] Table 6

[0113] Table 7

[0114] It is evident that by optimizing the modification method of the antisense chain, the present invention can significantly improve the silencing effect on PCSK9, and by optimizing the modification method of the justice chain, the silencing effect can be further enhanced.

[0115] Example 3 The hepatocyte-targeting compound Tri-GalNAc (the structure of which is shown in Formula I, and the relevant sequences are shown in Table 8) was conjugated and subjected to free uptake experiments in human primary hepatocytes. The siRNA-GalNAc samples were dissolved in 100 μL of enzyme-free sterile water to prepare a 10000 μM solution. 10 μL of the 10000 μM test solution was added to 90 μL of PMonH plating medium to dilute it to a 1000 μM solution as the working solution for the 1000 nM final concentration group. The 1000 μM test solution was then diluted with PMonH plating medium at 7 concentration points to make the final working solution concentrations 0.037, 0.11, 0.33, 1.1, 3.3, 10, 100, and 1000 nM. Human primary hepatocytes were removed from liquid nitrogen, thawed and revived at 37°C, washed with serum-containing PMonH plating medium, counted, and centrifuged. After removing the supernatant, the cells were diluted to 250 kJ / mL with fresh serum-containing PMonH plating medium. 90 μL of the diluted cell solution was then plated onto 96-well cell culture plates, resulting in 25 kJ / well. Prepared sample working solution was added to the cell solution to achieve final concentrations of 0.037, 0.11, 0.33, 1.1, 3.3, 10, 100, and 1000 nM. The plates were then incubated at 37°C for 48 hours with 5% CO2. After 48 hours, all culture medium was aspirated from the 96-well plates. The plates were washed with 1×PBS buffer, and 50 μL of prepared Cells to CT lysis buffer (as recommended by the manufacturer) was added and mixed. After standing for 10 min, 2.5 μL of stop solution was added to terminate the lysis for 2 min. RT-PCR was performed according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, catalog number: 4368814), with each reaction containing 10 μL of lysed liquid. Gene expression was quantified using real-time fluorescence PCR. The TaqMan probe for monkey PCSK9 was Mf03418189_m1, and the probe for the internal control gene (monkey PPIB) was Mf02802985_m1 (Thermo Fisher Scientific, Waltham, MA, USA). PCR conditions were 95°C for 20 seconds per cycle, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds. The real-time fluorescence PCR instrument was a QuantStudio™ 6 Pro real-time fluorescence quantitative PCR system (Thermo Fisher).

[0116] PCSK9 gene expression was calculated as 2^-ΔΔCt, with PPIB gene expression used as an internal control. PCSK9 gene silencing was calculated as a percentage of the control group (cells with only culture medium). The concentration of siRNA in each experimental group that reduced PCSK9 relative expression by 50% (IC50) was used. 50 The values ​​are shown in Table 9.

[0117] Table 8

[0118] Table 9

[0119] It is evident that the RNAi drug of the present invention has a far superior silencing effect on PCSK9 in primary monkey hepatocytes compared to existing superior drugs, and the preferred modification scheme of the present invention further demonstrates its efficacy advantages.

[0120] Example 4 To further verify the inhibitory effect of siRNA on PCSK9 expression, experiments were conducted in human PCSK9 transgenic mice. The siRNA-Tri-GalNAc compound (Seq ID No. 13 in Table 8) or PBS was subcutaneously injected into mice on day 0. Blood samples were collected on day 14, and plasma PCSK9 protein levels were measured using an ELISA (R&D Systems, catalog number DPC900, USA). The reduction effect was expressed as a percentage compared to pre-injection PCSK9 protein levels. The results are shown in Table 10 below.

[0121] Table 10

[0122] The results show that the RNAi drug of the present invention has a significantly better PCSK9 silencing effect than existing superior drugs in human PCSK9 transgenic mice, and the preferred modification scheme of the present invention further demonstrates its superior efficacy. Among them, SNK-680018 exhibits a significantly better PCSK9 silencing effect.

[0123] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. An RNAi reagent comprising a sense strand and an antisense strand; in, The antisense strand contains the nucleotide sequence from position 1 to position 21 of 5'-ACAAAAGCAAAACAGGUCUAGAA-3' (SEQ ID No. 1); The sense strand contains a nucleotide sequence that is at least partially complementary to the antisense strand; All nucleotides in the antisense strand are modified nucleotides and satisfy at least one of the following conditions I) to II): I) The antisense strand does not contain three consecutive identical chemical modifications at nucleotide positions 1 to 20 of the sequence shown in SEQ ID No. 1; II) The antisense strand is at position 2' of the first nucleotide of the sequence shown in SEQ ID No.

1. Fluoronucleotides; Preferably, the nucleotides modified at other positions in the antisense strand are each independently selected from 2' O Methylnucleotide, 2' Fluoronucleotides.

2. The RNAi reagent according to claim 1, wherein, The antisense strand is located at the 2' position of the 4th and 5th nucleotides of the sequence shown in SEQ ID No.

1. Fluoronucleotide, the 6th nucleotide is at the 2' position. O Methyl nucleotide.

3. The RNAi reagent according to claim 1 or 2, wherein, The antisense strand is located at positions 2' and 14 of the sequence shown in SEQ ID No.

1. Fluoronucleotides; Preferably, the antisense strand is 2' at positions 2, 8, 10, 12, 14, 16, and 18 of the sequence shown in SEQ ID No.

1. Fluoronucleotides.

4. The RNAi reagent according to any one of claims 1 to 3, wherein, The positive strand contains the nucleotide sequence 5'-CUAGACCUGUUUUGCUUUUGU-3' (SEQ ID No. 2) or 5'-CUAGACCUGUTUUGCUUUUGU-3' (SEQ ID No. 3); all nucleotides in the positive strand are modified nucleotides, and the positive strand has a 2' position at the first and / or third nucleotide position of the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotide; the positive strand is at position 2' of the 11th nucleotide in the sequence shown in SEQ ID No.

3. Deoxythymidine; preferably, the nucleotides modified at other positions in the positive strand are each independently selected from 2' O Methylnucleotide, 2' Fluoronucleotides.

5. The RNAi reagent according to claim 4, wherein, The positive strand contains the nucleotide sequence 5'-CUAGACCUGUUUUGCUUUUGU-3' (SEQ ID No. 2), and the positive strand is 2' at the 11th nucleotide position of the sequence shown in SEQ ID No.

2. Fluoronucleotides.

6. The RNAi reagent according to claim 4 or 5, wherein, The positive strand is located at position 2' of the 10th nucleotide in the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides.

7. The RNAi reagent according to any one of claims 4 to 6, wherein, The positive strand is 2' at position 7 and / or 9 of the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides; preferably, the positive strand is independently selected from the 2' end at the 8th and 14th nucleotide positions of the sequence shown in SEQ ID No. 2 or 3. Fluoronucleotides, 2' O Methyl nucleotides, and all other modified nucleotides are 2' nucleotides. O Methyl nucleotide.

8. The RNAi reagent according to any one of claims 1 to 7, wherein, The last three nucleotides at the 5' end of the positive strand contain at least one phosphate thioester nucleotide bond. The last three nucleotides at the 5' end of the antisense strand contain at least one phosphate thioester nucleotide bond, and the last five nucleotides at the 3' end of the antisense strand contain at least one phosphate thioester nucleotide bond.

9. The RNAi reagent according to any one of claims 1 to 8, wherein, The 5' end and / or 3' end of the positive chain contain inverted base deletion adapters.

10. The RNAi reagent according to any one of claims 1 to 9, wherein, The 3' end of the antisense chain contains: i) at least one 2' Deoxythymidine; or ii) at least one 2' O Methyluridine.

11. The RNAi reagent according to claim 1, wherein, The antisense strand contains the nucleotide sequence shown in Table 1 for any of the antisense strands: Preferably, the positive strand contains a nucleotide sequence as shown in any of the positive strands in Table 1: Preferably, the RNAi reagent contains any of the siRNAs shown in Table 1.

12. The RNAi reagent according to claim 1, wherein, The antisense strand contains the nucleotide sequence from position 1 to position 21 of 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13); Preferably, the sense strand contains the nucleotide sequence 5'-CfsusAfgacCfuGfUfUfuugcuuuugu-3' (SEQ ID No. 4), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13); or The sense strand contains the nucleotide sequence 5'-CfsusAfgacCfuGfUfUfuuGfcuuuugu-3' (SEQ ID No. 5), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13); or The sense strand contains the nucleotide sequence 5'-CfsusAfgacCfUfGfUfUfuuGfcuuuugu-3' (SEQ ID No. 6), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13); or The sense strand contains the nucleotide sequence 5'-CfsusAfgacCfuGfudTuugcuuuugu-3' (SEQ ID No. 7), and the antisense strand contains the nucleotide sequence 5'-AfsCfsaAfAfagCfaAfaAfcAfgGfuCfuagsasa-3' (SEQ ID No. 13); In each sequence, nucleotides represented by lowercase letters indicate that the nucleotide is 2'. O Methyl nucleotide; f indicates that the nucleotide adjacent to it on the left is 2'. Fluoronucleotides; 's' indicates that two adjacent nucleotides are linked by a phosphothioester bond.

13. The RNAi reagent according to any one of claims 1 to 12, wherein, The RNAi reagent further comprises at least one ligand, preferably, the at least one ligand being linked to one or more nucleotides of the antisense or sense strand, the nucleotides being selected from 5' terminal nucleotides, 3' terminal nucleotides, and / or any intra-strand nucleotide; preferably, the ligand is a GalNAc derivative; more preferably, the ligand is one or more GalNAc derivatives linked by a single-stranded, double-stranded, or triple-stranded branched ligand; even more preferably, the RNAi reagent comprises a compound with the following structure: In the formula, Nu represents a nucleic acid composed of the sense strand and the antisense strand.

14. A pharmaceutical composition comprising the RNAi reagent according to any one of claims 1 to 13.

15. A method for inhibiting the expression of PCSK9 in cells, the method comprising: The cells are contacted with the RNAi reagent of any one of claims 1 to 13 or the pharmaceutical composition of claim 14 to inhibit the expression of PCSK9 in the cells.

16. Use of the RNAi reagent according to any one of claims 1 to 13 or the pharmaceutical composition according to claim 14 in any of the following aspects: 1) Treatment and / or prevention of diseases associated with PCSK9; 2) Prepare medicines for the treatment and / or prevention of diseases related to PCSK9.

17. The use according to claim 16, wherein, The disease in question is: (i) Diseases associated with increased or elevated PCSK9 levels; or (ii) Diseases that will benefit from reduced PCSK9 expression.

18. The use according to claim 16, wherein, The disease is a cardiovascular disease, preferably selected from one or more of hyperlipidemia, hypercholesterolemia, coronary heart disease, myocardial infarction, stroke, and atherosclerosis.

19. The use according to any one of claims 16 to 18, wherein, The RNAi reagent or the pharmaceutical composition is administered to the subject via subcutaneous, intravenous, and / or intramuscular administration.

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