Nucleic acid targeting blood coagulation factor XI and application thereof

Abstract

The invention relates to the technical field of biology, in particular to nucleic acid targeting a blood coagulation factor XI and application of the nucleic acid. The nucleic acid provided by the invention can effectively reduce the blood coagulation factor XI level, and can effectively inhibit the formation of thrombus, so that various diseases caused by thrombus, such as pulmonary embolism, venous embolism, deep vein embolism, myocardial infarction, stroke and the like, can be prevented and / or treated.

Description

[0001] Cross - reference to related applications

[0002] This invention claims the priority of a Chinese patent application with the application number 202410368288.1 filed on March 28, 2024, the entire content of which is incorporated herein by reference. Technical field

[0003] This invention relates to the field of biotechnology, and specifically, to nucleic acids targeting coagulation factor XI and their uses. Background art

[0004] The blood coagulation mechanism is an indispensable function of human blood circulation. Normal blood coagulation function can prevent blood loss. The blood coagulation reaction generally starts from two pathways, the extrinsic and intrinsic pathways. The extrinsic pathway is also called the tissue factor pathway, which is activated by tissue damage - induced tissue factor (TF or Factor III), binds to coagulation factor VIIa, and then activates factor X into Xa. The intrinsic pathway is also called the contact activation pathway, which starts with the conversion of factor XII to XIIa caused by surface or vascular endothelial damage. Subsequently, XIIa converts factor XI to XIa, and XIa converts factor IX to IXa, which in turn activates factor X into Xa. From here, the two activation pathways enter the common pathway of the blood coagulation reaction. Xa binds to cofactor Va to form the prothrombinase complex (also known as thromboplastin), which promotes the activation of prothrombin (factor II) into thrombin (factor IIa). Thrombin then converts fibrinogen (factor I) into fibrin (factor Ia), and also activates factor XIII and converts it into XIIIa, further promoting fibrin cross - linking to form a thrombus.

[0005] Abnormalities in the blood coagulation mechanism can lead to various diseases. For example, deficiencies in coagulation factors VIII and IX can cause hemophilia, while thrombus formation and resulting vascular embolism are the root causes of various diseases such as pulmonary embolism, deep vein thrombosis, myocardial infarction, stroke, etc. Clinically commonly used antithrombotic therapies such as heparin, low - molecular - weight heparin, or warfarin all have limitations and are prone to treatment - related bleeding. The formation of many embolisms is considered to be caused by the activation of the blood coagulation pathway after surgery. In the study of people with factor XI deficiency (hemophilia C), it was found that generally, the bleeding tendency of this population is no different from that of normal people, and there is only a bleeding tendency during surgery and severe trauma. This makes factor XI a potential target for treating thrombus formation.

[0006] RNA interference (RNAi) refers to a phenomenon of highly conserved homologous mRNA efficient and specific degradation induced by small interfering RNA (siRNA) during the process of evolution. At the same time, RNAi drugs also have the advantage of a longer drug effect time compared to antibodies. Therefore, it is of great significance to research and develop siRNA targeting coagulation factor XI. Summary of the Invention

[0007] The object of the present invention is to overcome the problems existing in the prior art and provide a new nucleic acid targeting coagulation factor XI and its uses.

[0008] The first aspect of the present invention provides a nucleic acid, which comprises a sense strand and an antisense strand. Among them, the sense strand contains at least 15 consecutive nucleotides, and compared with any at least 15 consecutive nucleotides in the sequence shown in any of SEQ ID No. 1-121, the difference does not exceed 3 nucleotides; the antisense strand contains at least 15 consecutive nucleotides, and compared with any at least 15 consecutive nucleotides in the nucleotide sequence shown in any of SEQ ID No. 121-240, the difference does not exceed 3 nucleotides.

[0009] The second aspect of the present invention provides a targeted drug delivery system, which comprises a targeting group, a linking group, and the nucleic acid as described above linked to the targeting group through the linking group.

[0010] The third aspect of the present invention provides an in vitro cell containing the nucleic acid.

[0011] The fourth aspect of the present invention provides a pharmaceutical composition, which contains the nucleic acid or the targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

[0012] The fifth aspect of the present invention provides a method for inhibiting the expression of coagulation factor XI in cells, which comprises: contacting the cells with the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above to inhibit the expression of coagulation factor XI in the cells.

[0013] The sixth aspect of the present invention provides the uses of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above in any of the following aspects: 1) treating and / or preventing coagulation-related diseases; 2) preparing a drug for treating and / or preventing coagulation-related diseases.

[0014] The nucleic acid of the present invention can effectively reduce the level of coagulation factor XI and effectively inhibit the formation of thrombus, thereby being able to prevent and / or treat various diseases caused by thrombus, such as pulmonary embolism, venous embolism, deep vein thrombosis, myocardial infarction, stroke, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0016] Figure 1 It shows the remaining serum F11 levels in human FXI transgenic mice on the 7th day after subcutaneous injection of different targeted drug delivery systems in the examples of the present invention.

[0017] Figure 2 It shows the tracking observation results of the remaining serum F11 levels in human FXI transgenic mice after subcutaneous injection of different modified drugs in the examples of the present invention.

[0018] Figure 3 It shows the activity verification results of SN-685299 in cynomolgus monkeys in the examples of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The following will detail the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention, and are not intended to limit the present invention. Those skilled in the art can make various modifications and changes to the present invention without departing from the scope or spirit of the present invention. For example, the features described or illustrated as part of one embodiment can be used in another embodiment to produce a further embodiment.

[0020] TERMINOLOGY DESCRIPTION

[0021] Unless otherwise specified, the meanings of all terms (including technical and scientific terms) used to disclose the present invention are the same as those commonly understood by those of ordinary skill in the art to which the present invention pertains. Through further guidance, the following definitions are used to better understand the teachings of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] As used herein, the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The any and all combinations include combinations of any two related listed items, any more than two related listed items, or 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, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution connected by "logical AND").

[0023] The terms "comprising", "including", and "containing" used in the present invention are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0024] The numerical ranges represented by endpoints in the present invention include all the numerical values and fractions included within the range, as well as the recited endpoints.

[0025] In the present invention, the concentration values involved include fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, for 2%, a fluctuation within the range of ±0.1% is allowed. For larger numerical values or those that do not require overly precise control, a greater fluctuation is also allowed for their meaning. For example, for 100 mM, fluctuations within the ranges of ±1%, ±2%, ±5%, etc. are allowed. Regarding the molecular weight, a fluctuation of ±10% is allowed for its meaning.

[0026] In the present invention, descriptions such as "a plurality of" and "a variety of", without special limitations, refer to a quantity greater than or equal to 2.

[0027] In the present invention, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open-ended technical solution containing the listed features.

[0028] In the present invention, "preferred", "better", "more preferred", and "preferably" are only used to describe the embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of the present invention.

[0029] In the present invention, "optionally", "optional", "option", "alternatively", "alternative", "alternate" mean having or not having, that is, any one selected from two alternative options of "having" or "not having". If "optionally" or "alternatively" appears multiple times in a technical solution, without special instructions, and without contradictions or mutual restrictions, each "optionally" or "alternatively" is independent.

[0030] In the present invention, the term "nucleic acid" refers to a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules, and the oligonucleotide molecules can degrade or inhibit (e.g., degrade or inhibit under appropriate conditions) the translation of messenger RNA (mRNA) transcripts of target mRNA in a sequence-specific manner. The nucleic acid can act through the RNA interference mechanism (i.e., induce RNA interference by interacting with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells), or through any alternative mechanism or pathway. The defined scope of nucleic acids including sense strands and antisense strands disclosed herein includes but is not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates.

[0031] In the present invention, when referring to "the antisense strand (sense strand) contains at least 15 consecutive nucleotides, and compared with any at least 15 consecutive nucleotides in the reference sequence (such as the sequence shown in SEQ ID No. X or the nucleotide sequence at positions 1 to 21 of the shown sequence), the difference does not exceed 3 nucleotides", the alignment situations involved include the alignment with at least 15 consecutive nucleotides starting from any position (such as the 1st, 2nd, 3rd,..., 7th, or 8th position) in the reference sequence. As an example, in some embodiments, the antisense strand (sense strand) contains 21 consecutive nucleotides, where there are 1, 2, or 3 differences between the nucleotide sequence at positions 1 to 15 and the nucleotide sequence at positions 2 to 16 of the reference sequence, and the nucleotides at positions 16 to 20 in the antisense strand (sense strand) are the same or different from the nucleotide sequence at positions 17 to 21 of the reference sequence. Such sequences are also within the scope defined by the present invention. In some embodiments, the antisense strand (sense strand) contains 21 consecutive nucleotides, where the nucleotide sequence at positions 1 to 15 is the same as the nucleotide sequence at positions 1 to 15 of the reference sequence, and the nucleotides at positions 16 to 21 in the antisense strand (sense strand) are the same or different from the nucleotide sequence at positions 16 to 21 of the reference sequence. Such sequences are also within the scope defined by the present invention.

[0032] In the present invention, when referring to descriptions related to sequence alignment, the "difference" or "difference by" mentioned includes one or several of substitution, insertion, and deletion.

[0033] In the present invention, "Factor XI (FXI or F11)" is also known as plasma prekallikrein, and is a protein involved in the blood coagulation process. It plays an important role in the coagulation cascade, especially in the intrinsic pathway. When a blood vessel is damaged, a series of enzymatic reactions are activated, ultimately leading to the formation of fibrin, which helps stop bleeding and heal wounds.

[0034] In the present invention, when referring to the expression of a given gene, the terms "silence", "reduce", "inhibit", "down-regulate" or "knockdown" mean that, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein or protein subunit translated from mRNA in a cell, cell population, tissue, organ or subject in which the gene is transcribed, compared to a second cell, cell population, tissue, organ or subject not so treated, when the cell, cell population, tissue, organ or subject is treated with the nucleic acid described herein, the expression of the gene is reduced.

[0035] In the present invention, "fully complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, 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 can include all or part of the first nucleotide sequence or the second nucleotide sequence.

[0036] In the present invention, "partially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 70% but not all 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 can include all or part of the first nucleotide sequence or the second nucleotide sequence.

[0037] In the present invention, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequence molecules, at least 85% but not all 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 can include all or part of the first nucleotide sequence or the second nucleotide sequence.

[0038] In the present invention, when referring to "at least partially complementary", it 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 fully complementary.

[0039] In the present invention, the term "treatment" refers to a method or step taken to provide alleviation or reduction in the number, severity, and / or frequency of one or more disease symptoms in a subject. The treatment may include prevention, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more disease symptoms in the subject.

[0040] In the present invention, the term "link" means the joining of two compounds or molecules by a covalent bond. Unless otherwise specified, as used herein, the term "link" may refer to a connection between a first compound and a second compound with or without any intervening atom or group of atoms.

[0041] Nucleic acid

[0042] The present invention provides a (modified or unmodified) nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides have no more than 0, 1, 2, or 3 nucleotide differences compared to any at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in the sequence shown in any of SEQ ID Nos. 1 to 121; the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides have no more than 0, 1, 2, or 3 nucleotide differences compared to any at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the nucleotide sequence shown in any of SEQ ID Nos. 121 to 240.

[0043] In some embodiments, the antisense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides have no more than 0, 1, 2, or 3 nucleotide differences compared to any at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in the nucleotide sequence of positions 1 to 21 shown in any of SEQ ID Nos. 121 to 240.

[0044] In some embodiments, the antisense strand has 15 to 30 (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides (bases).

[0045] In some embodiments, the sense strand has 15 to 30 (such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides (bases).

[0046] In the present invention, the sense strand and the antisense strand may have the same length or different lengths.

[0047] In specific implementations, those skilled in the art can combine the sequences provided in the present invention in consideration of the complementarity of the sense strand and the antisense strand to obtain the combined nucleic acid (such as siRNA).

[0048] In a preferred embodiment of the present invention, as shown in Table 1, the nucleic acid is selected from at least one of siRNA-1 with a sense strand sequence of SEQ ID No. 1 and an antisense strand sequence of SEQ ID No. 121, siRNA-2 with a sense strand sequence of SEQ ID No. 2 and an antisense strand sequence of SEQ ID No. 122, siRNA-3 with a sense strand sequence of SEQ ID No. 3 and an antisense strand sequence of SEQ ID No. 123, siRNA-4 with a sense strand sequence of SEQ ID No. 4 and an antisense strand sequence of SEQ ID No. 124, siRNA-5 with a sense strand sequence of SEQ ID No. 5 and an antisense strand sequence of SEQ ID No. 125..., siRNA-118, siRNA-119, and siRNA-120.

[0049] In some preferred embodiments, the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides have a difference of no more than 3 nucleotides compared to any at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in the sequences shown in any of SEQ ID No. 125, 126, 127, 129, 165, 166; the sense strand contains a nucleotide sequence that is at least partially complementary (such as partially complementary, substantially complementary, or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA has a significantly better inhibitory effect on factor XI.

[0050] In some preferred embodiments, the sense strand contains at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, and the at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides differ by no more than 3 nucleotides from any of at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides in the sequence shown in any of SEQ ID No.5, 6, 7, 9, 45, 46.

[0051] In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:7 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:127 by 0, 1 or 2 nucleotides.

[0052] In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:9 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:129 by 0, 1 or 2 nucleotides.

[0053] In some preferred embodiments, the sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:45 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:165 by 0, 1 or 2 nucleotides.

[0054] All nucleotide groups in the above nucleic acids can be unmodified or can contain at least one modified nucleotide group, and the modification can be on any nucleotide.

[0055] In some embodiments, the sense strand and the antisense strand can be partially complementary, substantially complementary or completely complementary to each other.

[0056] In some embodiments, when the sequence identity of the sense strand or the antisense strand in the nucleic acid is less than 100% or differs by more than 1 nucleotide from the corresponding sequence mentioned in the present invention, it still has an inhibitory effect on factor XI that is similar (such as still having an efficacy equivalent to 80-120%, 85-115% or 90-110% of the corresponding sequence) or equivalent (such as still having an efficacy equivalent to 95-105% of the corresponding sequence) to the corresponding sequence. For example, replace the two bases at the 3' end of the antisense strand (such as any of the sequences shown in SEQ IDNo.121-240) with UU, AA, CU, UC, AG, CC, GG or UG, etc., or any combination of two nucleic acids. Such nucleic acid sequences also fall within the protection scope of the present invention.

[0057] In some preferred embodiments, the nucleic acid has an inhibition efficiency against factor XI of not less than 50% (such as 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0058] Table 1

[0059]

[0060]

[0061]

[0062]

[0063] In Table 1, the first column indicates the position of the first base of the target gene in the coding sequence of human factor XI, and so on; the numbers in the third and fifth columns represent the sequence numbers. For example, "1" represents SEQ ID No.1. Among them, the reference sequence of the target gene is the coding sequence NM_000128.4 of human factor XI.

[0064] In the technical solutions of the present invention regarding the above-mentioned naked sequences (i.e., unmodified sequences), the effect advantages do not depend on the modification method or the choice of the targeting vector. The applicable modification schemes and further preferred modification schemes are introduced below.

[0065] In some embodiments, the nucleic acid contains nucleotide groups as basic structural units, and the nucleotide groups contain phosphate groups, ribose groups, and bases. Preferably, the nucleic acid contains at least one modified nucleotide group. The nucleic acid containing the modified group has an inhibition efficiency against factor XI of not less than 50% (such as 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%).

[0066] In some embodiments, the modified nucleotide group is a nucleotide group in which the phosphate group and / or the ribose group is modified. The modified site can be at least one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th nucleotides on the sense strand and / or the antisense strand, specifically at the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th positions.

[0067] In some embodiments, the modification of the phosphate group refers to the modification of the oxygen in the phosphate group, including phosphorothioate modification and boranophosphate modification, etc. As shown in the following formula, sulfur, borane, amino group, alkyl group or alkoxy group are respectively used to replace the oxygen in the phosphate group. These several modifications can all stabilize the structure of the nucleic acid and maintain high specificity and high affinity of base pairing.

[0068]

[0069]

[0070] In the above structural formula, BASE represents the base A, U, C, G, or T. X can be oxygen (O) or sulfur (S). R can be the same or different in the above structure, such as: hydrogen (H), fluorine (F), methoxy (OME), or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, amino, cyanoethyl, acetyl, etc. R' and R'' can each independently be hydrogen (H), methyl (CH3), ethyl (CH2CH3), propyl (CH2CH2CH3), isopropyl (CH(CH3)2), allyl, propargyl, acyloxybenzyl, acyloxyethyl.

[0071] In some embodiments, the modification of the ribose group refers to the modification of the 2'-hydroxy group (2'-OH) in the ribose group. After introducing certain substituents such as methoxy or fluorine at the 2'-hydroxy position of the ribose group, the nucleic acid is not easily cleaved by ribonuclease, thereby increasing the stability of the nucleic acid and making the nucleic acid have stronger resistance to nuclease hydrolysis. The modification of the 2'-hydroxy group in the pentose of the nucleotide includes 2'-fluoromodification (such as 2'-arabino-fluoromodification), 2'-methoxy modification (2'-OME), 2'-methoxyethyl modification (2'-MOE), 2'-2,4-dinitrophenol modification (2'-DNP modification), 2',4'-constrained ethylmodification, 2'-amino modification, 2'-deoxy modification, BNA, acyclic nucleic acid modification, misaligned nucleic acid modification, L-type nucleic acid modification, etc. BNA (bridged nucleic acid in the inner ring) refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C 3'-endo sugar puckering bridged structure. Usually, this bridge is incorporated into the 2'- and 4'-positions of the ribose ring to provide 2',4'-BNA nucleotides, such as locked nucleic acid modification (LNA), 2',4'-constrained ethyl nucleic acid modification (ENA), and cET BNA. Acyclic nucleic acids are nucleotides formed by opening the sugar ring of nucleotides, such as unlocked nucleic acid (UNA) nucleotides and glycerol nucleic acid (GNA) nucleotides. Misaligned nucleic acid modification refers to the replacement of the 3',5'-phosphodiester bond with a 2',5'-phosphodiester bond. L-type nucleic acid modification refers to the replacement of the naturally occurring D-type nucleic acid with its mirror stereoisomer L-type nucleic acid.

[0072]

[0073]

[0074] Among them, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

[0075] In some embodiments, according to a particularly preferred embodiment of the present invention, wherein the nucleotide group containing uracil base or cytosine base in the sense strand of the RNAi reagent is the nucleotide group with modified ribose group, that is, the 2'-OH of the ribose group in the nucleotide group containing uracil base or cytosine base in the sense strand of the RNAi reagent is replaced by methoxy or fluorine. More preferably, dTdT can be connected to the 3' ends of both the sense strand and the antisense strand of the RNAi reagent; or, AA or UU or any combination of two nucleic acids (which can be but not limited to CC, GG or UG) can be connected to the 3' end of the antisense strand of the RNAi reagent, so that the sequence has a specific inducement for mRNA degradation. The RNAi reagent with the above modifications shows a more excellent in vivo inhibitory effect, and the above modifications can further reduce the immunogenicity of the RNAi reagent of the present invention in vivo.

[0076] The RNAi reagent of the present invention can also include the modification of connecting a monophosphate nucleotide to the 5' end of the antisense strand. Since the 5'-monophosphate at the terminal of the siRNA guide strand is important for RISC recognition. The phosphorylation of the 5'-hydroxyl plays a certain role in whether the siRNA can be effectively loaded onto Ago2 inside the cell. There is a hydrogen bond interaction between the 5'-monophosphate at the 5' end of the guide strand in the siRNA and Argonaute-2 (Ago2), thus ensuring the accurate positioning and precise cleavage of the mRNA target. The following are several common derivatives of 5'-monophosphate nucleotides, and such derivatives of phosphonucleotides have been proven to have certain stability in biological metabolic media and play a certain role in promoting the loading of the siRNA guide strand into Ago2 inside the cell (Nucleic Acids Research, 2015, 43, 2993–3011). According to the RNAi reagent of the present invention, preferably, trans-vinyl phosphate (VP) is the first choice, and it can also include derivatives of monophosphate nucleotides other than those described above.

[0077]

[0078]

[0079] In the above structure, BASE represents the base A, U, C, G or T. R in the above structure can be the same or different, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

[0080] In the present invention, has the same meaning, referring to a chemical element X connected to any one or more groups.

[0081] In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified internucleoside bond.

[0082] In some embodiments, the modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-cyclic nucleotide analog, locked nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methyl nucleotide, inverted 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, nucleotide containing vinyl phosphonate, nucleotide containing cyclopropyl phosphonate, and 3'-O-methyl nucleotide; the modified nucleotide is further preferably selected from one or two of 2'-O-methyl nucleotide and 2'-fluoro nucleotide.

[0083] In some embodiments, the modified internucleoside bond is preferably selected from one or more of phosphorothioate internucleoside bond and methylphosphonate internucleoside bond. In some embodiments, the modified internucleoside bond is further preferably selected from one or more of phosphorothioate monoester internucleoside bond and phosphorothioate diester internucleoside bond.

[0084] In some preferred embodiments, the antisense strand is 2'-fluoro nucleotide at the nucleotide positions 2, 6, 14, and 16 and at least one (such as 1, 2, 3, or 4) of the nucleotide positions selected from 3, 4, 8, and 23 in the nucleotide sequence shown in any one of SEQ ID No. 121 - 240, and 2'-O-methyl nucleotide at other positions. Compared with the known modification methods, the above antisense strand modification scheme is further beneficial to enhancing the inhibitory effect of the nucleic acid on factor XI.

[0085] In some further preferred embodiments, the antisense strand is 2'-fluoro nucleotide at the nucleotide positions 2, 6, 8, 14, 16, and 23 in the nucleotide sequence shown in any one of SEQ ID No. 121 - 240, and 2'-O-methyl nucleotide at other positions.

[0086] In some preferred embodiments, the sense strand has 2'-fluoro nucleotides at nucleotide positions 7, 9, 10, and 11 and at least one (such as 1 or 2) of the nucleotide positions selected from 2 and 13 of the nucleotide sequence shown in any one of SEQ ID No. 1 to 120, and 2'-O-methyl nucleotides at other positions. Compared with known modification methods, the above sense strand modification scheme further facilitates enhancing the inhibitory effect of the nucleic acid on factor XI.

[0087] In some further preferred embodiments, the sense strand has 2'-fluoro nucleotides at nucleotide positions 2, 7, 9, 10, and 11 of the nucleotide sequence shown in any one of SEQ ID No. 1 to 120, and 2'-O-methyl nucleotides at other positions.

[0088] In some embodiments, the last 2 to 4 (such as 2, 3, or 4) nucleotides at the 5'-end and / or 3'-end of the antisense strand contain phosphorothioate internucleotide linkages, and the last 2 to 4 (such as 2, 3, or 4) nucleotides at the 5'-end and / or 3'-end of the sense strand contain phosphorothioate internucleotide linkages.

[0089] In some specific embodiments, the last 3 nucleotides at the 5'-end and 3'-end of the antisense strand contain phosphorothioate internucleotide linkages, and the last 3 nucleotides at the 5'-end of the sense strand contain phosphorothioate internucleotide linkages.

[0090] In some embodiments, the antisense strand contains a nucleotide sequence that differs from the antisense strand shown in any one of Table 2 or Table 3 by 0, 1, or 2 nucleotides.

[0091] In some embodiments, the sense strand contains a nucleotide sequence that differs from the sense strand shown in any one of Table 2 or Table 3 by 0, 1, or 2 nucleotides.

[0092] In some embodiments, the nucleic acid contains a duplex shown in any one of Table 2 or Table 3.

[0093] In some preferred embodiments, the nucleic acid contains a duplex selected from any one of SN-255886, SN-255887, SN-255888, SN-255880, SN-255826, SN-255827, SN-256683, SN-256684, SN-256685, SN-255295, SN-255296, SN-255297, SN-255298, SN-255299.

[0094] Table 2

[0095]

[0096]

[0097]

[0098]

[0099] Table 3

[0100] Number Sense strand (5’-3’) Antisense strand (5’-3’) SN-255888 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-256683 gsCfsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-256684 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgUfcuuugUfuGfcaagcsusu SN-256685 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsusUf SN-255888 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-255295 gsCfsuugcAfaCfAfAfagacauuuau asUfsAfaaUfgUfcuuugUfuGfcaagcsusUf SN-255296 gscsuugcAfaCfAfAfaGfacauuuau asUfsAfaaUfgUfcuuugUfuGfcaagcsusu SN-255297 gsCfsuugcAfaCfAfAfagacauuuau asUfsAfaaUfgUfcuuugUfuGfcaagcsusu SN-255298 gscsuugcAfaCfAfAfagacauuuau asUfsAfaaUfgUfcuuugUfuGfcaagcsusUf SN-255299 gsCfsuugcAfaCfAfAfagacauuuau asUfsaaaUfgUfcuuugUfuGfcaagcsusUf

[0101] In each modified sequence of the present invention, nucleotides represented by lowercase letters represent that the nucleotide is a 2'-O-methyl nucleotide; f represents that one nucleotide adjacent to its left is a 2'-fluoro nucleotide; s represents that the two nucleotides adjacent to the left and right are connected by a phosphorothioate internucleotide bond.

[0102] The nucleic acid of the present invention can be obtained by conventional methods in the art, such as by solid-phase synthesis and liquid-phase synthesis. Commercial custom services for solid-phase synthesis are available, so it can be obtained through commercial purchase. The modified nucleotide groups can be introduced by nucleotide monomers with corresponding modifications.

[0103] Based on the nucleic acid (siRNA) synthesized as above, the present invention can further construct an shRNA expression plasmid having the same or similar function as the above nucleic acid. The method for constructing this expression plasmid is well known to those skilled in the art and will not be elaborated here.

[0104] Targeted drug delivery system

[0105] The present invention also provides a targeted drug delivery system, which includes a targeting group, a linking group, and the nucleic acid as described above connected to the targeting group through the linking group.

[0106] Combined with common knowledge in the art, when the nucleic acid (siRNA) of the present invention is applied to different targeted drug delivery systems, it has excellent inhibitory effects. In other words, the effect advantages of the naked sequence and the modified sequence in the present invention do not depend on the choice of the targeting vector. To further improve the bioavailability and therapeutic effect of siRNA, the present invention has also optimized the targeted drug delivery system and obtained the following technical solutions.

[0107] In some specific embodiments, the targeting group can further improve the targeting of small nucleic acids and can be provided by monosaccharides (such as glucose, mannose, allose, altrose, galactose, galactosamine, N-acetylgalactosamine, talose, fructose, idose, etc.) and / or polypeptides (such as proteins, monoclonal antibodies, nanobodies).

[0108] In some specific embodiments, the linking group may be selected from -O-[CH2CH2O]n-, -[CH2]m-CONH-[CH2]nO-, -O-[CH2CH2O]m-CONH-[CH2]nO-, -O-[CH2]m-CONH-[CH2H2O]nO-. Wherein, m and n may each independently be an integer from 1 to 10.

[0109] In some embodiments, the targeted drug delivery system includes a ligand and the nucleic acid linked to the ligand, and the ligand is linked to one or more of the 5'-end of the antisense strand, the 3'-end of the antisense strand, the 5'-end of the sense strand, and the 3'-end of the sense strand.

[0110] In some preferred embodiments, the ligand is a GalNAc derivative.

[0111] In some preferred embodiments, the ligand is one or more GalNAc derivatives linked by a single-stranded, double-stranded or triple-stranded branched linker.

[0112] In some further preferred embodiments, the RNAi reagent contains a compound having the following structural formula I:

[0113]

[0114] In the formula, Nu represents the duplex. The targeted drug delivery system can improve the cell penetration ability of the nucleic acid drug (Nu), enhance its stability in cells, and has a simple preparation process and strong practicability by virtue of its structural characteristics on the left side.

[0115] In specific implementation, the ligand moiety can be contacted with a nucleotide monomer or a nucleic acid linked to a solid support in the presence of a coupling reaction condition and a coupling reagent, so that the compound moiety is linked to the nucleic acid through a coupling reaction.

[0116] cell

[0117] The present invention also provides an ex vivo cell containing the nucleic acid.

[0118] In some embodiments, the cell can be used for gene function research, disease model research, drug screening and other purposes.

[0119] In some embodiments, the cell will not develop into an animal individual. In some specific embodiments, the cell can be a microbial cell or an animal cell, but the animal cell is not an embryonic stem cell of an animal and cells at various formation and development stages thereof (such as germ cells, fertilized egg cells, etc.).

[0120] Drug composition

[0121] The present invention also provides a pharmaceutical composition, which contains the nucleic acid or the targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

[0122] The pharmaceutical composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier by conventional methods. For example, the pharmaceutical composition can be an injection solution. The injection solution can be used for subcutaneous, intramuscular or intravenous injection.

[0123] For the pharmaceutical composition according to the present invention, there is no particular requirement for the amounts of the nucleic acid or the targeted drug delivery system and the pharmaceutically acceptable carrier. Generally, relative to 1 part by weight of the nucleic acid (or 1 part by weight of the targeted drug delivery system calculated as nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100000 parts by weight (such as 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 values).

[0124] For the pharmaceutical composition according to the present invention, the pharmaceutically acceptable carrier can be various carriers conventionally used in the art. For example, it can include at least one of a pH buffer, a protecting 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 protecting agent can be at least one of inositol, sorbitol and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protecting agent can be 0.01-30% by weight (such as 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 values). The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmoles per kilogram. Those skilled in the art can determine the content of the osmotic pressure regulator according to the required osmotic pressure.

[0125] According to a preferred embodiment of the present invention, the pharmaceutically acceptable carrier is a liposome. The liposome can be any liposome capable of encapsulating nucleic acid, and its diameter can be 25-1000 nm, and can include but not limited to cholesterol and its analogs or derivatives.

[0126] The dosage of the pharmaceutical composition of the present invention can be a conventional dosage in the art, and the dosage can be determined according to various parameters, especially according to the age, weight and gender of the subject. For example, for female mice at 3-4 months of age with a weight of 25-30 g, based on the amount of the nucleic acid in the pharmaceutical composition, the dosage of the pharmaceutical composition can be 0.01-100 mg / kg body weight, preferably 1-10 mg / kg body weight.

[0127] Method and Use

[0128] The present invention also provides a method for inhibiting the expression of coagulation factor XI in cells, which comprises: contacting the cells with the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above to inhibit the expression of coagulation factor XI in the cells.

[0129] In some embodiments, the cells are in a subject, for example, a human subject, such as a subject suffering from a coagulation factor XI-related disease, or a subject in need of preventing the risk of a coagulation factor XI-related disease.

[0130] In some embodiments, the cells are located in vitro. The method is based on research purposes or for constructing an animal model.

[0131] In some embodiments, contacting the cells with the RNAi reagent or the pharmaceutical composition inhibits the expression of coagulation factor XI by at least 50%, 60%, 70%, 80%, 90%, 95% (for example, compared with the expression level of coagulation factor XI before the cells first contact the RNAi reagent or the pharmaceutical composition; for example, before administering the first dose of the RNAi reagent or the pharmaceutical composition to the subject). In certain embodiments, inhibiting the expression of coagulation factor XI reduces the level of coagulation factor XI protein in the serum sample of the subject by at least 50%, 60%, 70%, 80%, 90% or 95%, for example, compared with the expression level of coagulation factor XI before the cells first contact the RNAi reagent or the pharmaceutical composition.

[0132] The present invention also provides the use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above in the treatment and / or prevention of coagulation-related diseases. That is, a method for treating and / or preventing coagulation-related diseases, which comprises: administering the nucleic acid, the targeted drug delivery system or the pharmaceutical composition to a subject.

[0133] The present invention also provides the use of the nucleic acid, the targeted drug delivery system or the pharmaceutical composition as described above in the preparation of a drug for treating and / or preventing coagulation-related diseases.

[0134] In some embodiments, the disease is: (i) a disease associated with enhanced or elevated coagulation factor XI; or (ii) a disease that would benefit from reduced expression of coagulation factor XI.

[0135] In some embodiments, the disease is selected from diseases associated with coagulation disorders (such as an increased risk of thrombosis).

[0136] In some embodiments, the disease is selected from at least one of pulmonary embolism, venous embolism, deep vein thrombosis, myocardial infarction, and stroke.

[0137] In the present invention, the subject can be a mammal, including a primate (such as a human, a non-human primate, for example, a monkey and a chimpanzee), a non-primate (such as a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird. In some embodiments, the subject is preferably a primate, more preferably a human.

[0138] In some embodiments, administration can be via multiple routes, depending on whether local treatment or systemic treatment is required. The dosage can be referred to as described above and will not be elaborated herein.

[0139] In some embodiments, administration can be local (such as a transdermal patch), pulmonary, such as inhalation or insufflation via a powder or a spray, including via a nebulizer; intratracheal, nasal, epidermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subepidermal, such as via an implant device; or intracranial, such as via intracerebral, intrathecal, or intraventricular administration.

[0140] In some embodiments, the nucleic acid, the targeted drug delivery system, or the pharmaceutical composition is administered to the subject by subcutaneous administration, intravenous administration, and / or intramuscular administration.

[0141] Examples

[0142] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present invention is preferably referred to, and it can also be in accordance with the experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or the conditions recommended by the manufacturer.

[0143] In the following specific examples, for the measurement parameters of the raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0144] Example 1

[0145] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 10 4 Hep3B (Procell, Cat#CL-0102) cells into a 96-well cell culture dish and incubate overnight in a cell incubator at 37 °C and 5% CO2. Add RNAiMAX (1.5 μL / well) and small interfering nucleic acid (siRNA) with the sequences shown in Table 2 into Opti-MEM medium, and add it to the cell culture wells so that the final concentration in each well is 1 nM or 10 nM. Continue to culture for 48 hours in a cell incubator at 37 °C and 5% CO2. To extract RNA, aspirate the cell culture supernatant, rinse with PBS, and after aspiration, add 50 μL of the prepared lysis solution (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)), mix well, let stand for 10 min, and then add 2.5 μL of Stop solution to terminate for 2 min. Perform RT-PCR according to the recommendations of the High Capacity cDNA ReverseTranscription Kits (Thermo Fisher, catalog number: 4368814), with 10 μL of the lysed liquid in each reaction. Measure gene expression quantification by real-time fluorescence PCR. The TaqMan probe for human FXI is Hs01038035_m1, and the probe for the reference gene (human HPRT1) is Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions are 1 cycle of 95 °C for 20 seconds, and 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The real-time fluorescence PCR instrument is QuantStudio TM 6Pro real-time fluorescence quantitative PCR system (Thermo Fisher). The expression of the FXI gene is calculated as 2^-ΔΔCt, and the expression of the human HPRT1 gene is used as an internal reference. The expression level of the FXI gene is expressed as a relative value with the cell group containing only RNAiMAX as the control. The results are shown in Table 4.

[0146] Table 4 Silencing effect of F11 mRNA expression in Hep3B cells

[0147] Compound 1nM 10nM Compound 1nM 10nM Compound 1nM 10nM SN-255872 0.45 0.40 SN-255823 0.62 0.30 SN-255964 0.78 0.58 SN-255883 0.58 0.44 SN-255824 0.78 0.50 SN-255965 0.53 0.52 SN-255884 0.42 0.43 SN-255825 0.74 0.66 SN-255966 0.68 0.63 SN-255885 0.60 0.34 SN-255826 0.42 0.34 SN-255967 0.55 0.40 SN-255886 0.35 0.39 SN-255827 0.40 0.45 SN-255968 0.68 0.33 SN-255887 0.39 0.28 SN-255828 0.67 0.64 SN-255969 0.62 0.40 SN-255888 0.23 0.26 SN-255829 0.58 0.48 SN-255960 0.99 0.78 SN-255889 0.41 0.30 SN-255820 0.49 0.44 SN-255961 0.82 0.66 SN-255880 0.36 0.37 SN-255821 0.46 0.35 SN-255962 0.90 0.72 SN-255881 0.68 0.47 SN-255822 0.62 0.60 SN-255973 0.53 0.81 SN-255882 0.66 0.44 SN-255933 0.62 0.50 SN-255974 0.65 0.73 SN-255893 0.52 0.35 SN-255934 0.91 0.80 SN-255975 0.47 0.58 SN-255894 0.44 0.37 SN-255935 0.49 0.52 SN-255976 0.59 0.60 SN-255895 0.57 0.41 SN-255936 0.53 0.57 SN-255977 0.77 1.04 SN-255896 0.60 0.38 SN-255937 0.63 0.59 SN-255978 0.41 0.72 SN-255897 0.39 0.45 SN-255938 0.59 0.49 SN-255979 0.64 1.19 SN-255898 0.89 0.93 SN-255939 0.75 0.67 SN-255970 0.62 0.69 SN-255899 0.62 0.69 SN-255930 0.91 0.97 SN-255971 0.71 0.79 SN-255890 0.79 0.93 SN-255931 0.58 0.58 SN-255972 0.36 0.67 SN-255891 0.54 0.33 SN-255932 0.83 0.54 SN-255983 0.47 0.63 SN-255892 0.60 0.55 SN-255943 0.52 0.43 SN-255984 0.46 0.73 SN-255803 0.51 0.32 SN-255944 0.63 0.47 SN-255985 0.43 0.45 SN-255804 0.66 0.49 SN-255945 0.52 0.58 SN-255986 0.72 0.74 SN-255805 0.64 0.55 SN-255946 0.57 0.63 SN-255987 0.38 0.63 SN-255806 0.49 0.42 SN-255947 0.45 0.46 SN-255988 0.55 0.75 SN-255807 0.48 0.42 SN-255948 0.46 0.65 SN-255989 0.46 0.66 SN-255808 0.59 0.26 SN-255949 0.75 0.72 SN-255980 1.01 1.16 SN-255809 0.73 0.44 SN-255940 0.38 0.29 SN-255981 0.71 0.68 SN-255800 0.58 0.49 SN-255941 0.34 0.50 SN-255982 0.48 0.79 SN-255801 0.55 0.66 SN-255942 0.77 0.50 SN-255993 0.74 0.75 SN-255802 0.61 0.43 SN-255953 0.60 0.37 SN-255994 0.58 0.77 SN-255813 0.40 0.24 SN-255954 1.08 0.99 SN-255995 0.75 0.70 SN-255814 0.48 0.58 SN-255955 0.63 0.71 SN-255996 0.36 0.70 SN-255815 0.42 0.47 SN-255956 0.57 0.65 SN-255997 0.64 0.91 SN-255816 0.50 0.36 SN-255957 0.65 0.43 SN-255998 0.60 0.59 SN-255817 0.95 0.55 SN-255958 0.75 0.57 SN-255999 0.96 1.02 SN-255818 0.48 0.41 SN-255959 0.59 0.47 SN-255990 0.48 0.59 SN-255819 0.46 0.44 SN-255950 0.75 0.85 SN-255991 0.62 0.82 SN-255810 0.86 0.71 SN-255951 0.75 0.90 SN-255811 0.76 0.58 SN-255952 0.89 0.90 SN-255812 0.52 0.38 SN-255963 0.61 0.79

[0148] It can be seen that the siRNA containing the double-stranded sequence of the present invention has different degrees of inhibitory effects on the expression of FXI.

[0149] Example 2

[0150] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 10 4 Hep3B (Procell, Cat#CL-0102) cells into a 96-well cell culture dish and incubate overnight in a cell incubator at 37 °C and 5% CO2. Add RNAiMAX (1.5 μL / well) and the small interfering nucleic acid (siRNA) in Table 5 into Opti-MEM medium, and add it to the cell culture wells so that the final concentration in each well is 0.0137 nM, 0.0411 nM, 0.1235 nM, 0.3704 nM, 1.111 nM, 3.333 nM, and 10 nM. Continue to incubate in a cell incubator at 37 °C and 5% CO2 for 48 hours. To extract RNA, aspirate the cell culture supernatant, wash with PBS, aspirate again, add 50 μL of the prepared lysis solution (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)), mix well, let stand for 10 min, and then add 2.5 μL of Stop solution to terminate for 2 min. Perform RT-PCR according to the recommendations of the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher, catalog number: 4368814), with 10 μL of the lysed liquid in each reaction. Measure the gene expression quantification by real-time fluorescence PCR. The TaqMan probe for human FXI is Hs01038035_m1, and the probe for the internal reference gene (human HPRT1) is Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions are 1 cycle of 95 °C for 20 seconds, and 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The real-time fluorescence PCR instrument is QuantStudio TM 6 Pro real-time fluorescence quantitative PCR system (ThermoFisher). The FXI gene expression is calculated as 2^-ΔΔCt, and the human HPRT1 gene expression is used as an internal reference. The FXI gene expression level is expressed as a relative value compared to the cell group with only RNAiMAX as a control, and the IC50 value is calculated. The results are shown in Table 5.

[0151] Table 5 IC50 values of different siRNAs for knocking down F11 expression in Hep3B cells

[0152] Number IC50, nM Number IC50, nM SN-255886 0.0032 SN-255940 15.332 SN-255887 0.0491 SN-255813 0.1533 SN-255888 0.0025 SN-255826 0.0212 SN-255889 4.511 SN-255827 0.0899 SN-255880 0.005 SN-255821 14.079

[0153] Example 3

[0154] To further verify the activity of siRNA, siRNA with good activity in Hep3B cells was selected and conjugated with TriGalNAc (the structure of this compound is shown in Formula I). The sequences and connection methods of this targeted drug delivery system are shown in Table 6. On the 0th day, 3 mg / kg of the targeted drug delivery system shown in Table 6 or PBS (control group) was subcutaneously injected into different human FXI transgenic mice. On the 7th day, human FXI protein in the blood was tracked and observed. The results are as Figure 1 .

[0155] Table 6

[0156] Number Sense strand (5’-3’) Antisense strand (5’-3’) SN-685886 asgscgcuUfgCfAfAfcaaagacauu-TriGalNAc asAfsuGfuCfuuuguugCfaAfgcgcususu SN-685887 gscsgcuuGfcAfAfCfaaagacauuu-TriGalNAc asAfsaUfgUfcuuuguuGfcAfagcgcsusu SN-685888 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-685880 gsascaugAfaGfGfGfcauaaacuau-TriGalNAc asUfsaGfuUfuaugcccUfuCfaugucsusu SN-685826 gscsauuuUfaAfAfUfcaaucugaaa-TriGalNAc usUfsuCfaGfauugauuUfaAfaaugcsusu SN-685827 csasuuuuAfaAfUfCfaaucugaaau-TriGalNAc asUfsuUfcAfgauugauUfuAfaaaugsusu

[0157] Example 4

[0158] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 10 4 Hep3B (Procell, Cat#CL-0102) cells into a 96-well cell culture dish and incubate overnight in a cell incubator at 37 °C and 5% CO2. Add RNAiMAX (1.5 μL / well) and the small interfering nucleic acid (siRNA) shown in Table 7 to the Opti-MEM culture medium, and add it to the cell culture wells so that the final concentration in each well is 0.4 nM or 2 nM. Continue to culture in a cell incubator at 37 °C and 5% CO2 for 48 hours. To extract RNA, aspirate the cell culture supernatant, rinse with PBS, and after aspiration, add 50 μL of the prepared lysis solution (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)), mix well, let stand for 10 min, and then add 2.5 μL of Stop solution to terminate for 2 min. Perform RT-PCR according to the recommendations of the High Capacity cDNA ReverseTranscription Kits (Thermo Fisher, catalog number: 4368814), with 10 μL of the lysed liquid in each reaction. Use real-time fluorescence PCR to measure gene expression quantification. The TaqMan probe for human FXI is Hs01038035_m1, and the probe for the internal reference gene (human HPRT1) is Hs02800695_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions are 1 cycle of 95 °C for 20 seconds, and 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The real-time fluorescence PCR instrument is QuantStudio TM6 Pro Real-Time Fluorescent Quantitative PCR System (Thermo Fisher). The expression of the FXI gene was calculated by 2^-ΔΔCt, and the expression of the human HPRT1 gene was used as an internal reference. The expression level of the FXI gene was expressed as a relative value with the cell group with only RNAiMAX as the control. The results are shown in Table 8.

[0159] Table 7

[0160] Number Sense strand (5’-3’) Antisense strand (5’-3’) SN-255888 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-256581 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsgsc SN-256582 csusugcaAfcAfAfAfgacauuuauu asAfsuAfaAfugucuuuGfuUfgcaagsusu SN-256593 csgscuugCfaAfCfAfaagacauuua usAfsaAfuGfucuuuguUfgCfaagcgsusu SN-256594 csusugcaAfcAfAfAfgacauuuau asUfsaAfaUfgucuuugUfuGfcaagsusu SN-256595 gscsuugcAfaCfAfAfagacauuua usAfsaAfuGfucuuuguUfgCfaagcsusu SN-256596 csgscuugCfaAfCfAfaagacauuua asUfsaAfaUfgucuuugUfuGfcaagcsgsu SN-256597 gscsuugcAfaCfAfAfagacauuuau asAfsuAfaAfugucuuuGfuUfgcaagscsu SN-256598 gscsuugcAfaCfaAfagacauuuau asUfsaaaugucuuUfgUfuGfcaagcsgsc SN-256599 gscsuugcAfaCfaAfagacauuuau asUfsaaauGfucuuUfgUfuGfcaagcsgsc

[0161] Table 8

[0162] Serial number 0.4 nM 2 nM SN-255888 42.6 35.4 SN-256581 54.9 42.5 SN-256582 87.1 75.4 SN-256593 69.5 55.2 SN-256594 69 44.9 SN-256595 92.3 66.1 SN-256596 62 47.5 SN-256597 96.3 97.08 SN-256598 60.2 48.3 SN-256599 57.1 45.6

[0163] Example 5

[0164] To determine the changes in siRNA activity caused by modifications at different sites, the siRNAs in Table 9 were conjugated with the hepatocyte-targeting compound Tri-GalNAc (whose structure is shown in Formula I), and a free uptake experiment was conducted in primary monkey hepatocytes. The siRNA-GalNAc samples were dissolved in 100 μL of enzyme-free and sterile water to form 10,000 μM solutions. Then, 10 μL of the 10,000 μM test compound solution was added to 90 μL of PMonH plating medium and diluted to 1,000 μM solution as the working solution for the 1,000 nM final concentration group; the 1,000 μM test compound solution was further serially diluted 3-fold at 8 concentration points with PMonH plating medium to make the final working solution concentrations 0.5 μM, 1.4 μM, 4 μM, 12 μM, 37 μM, 111 μM, 333 μM, and 1,000 μM. Primary monkey hepatocytes were taken out from liquid nitrogen, thawed and recovered at 37 °C, rinsed, counted, and centrifuged with serum-containing PMonH plating medium. After removing the supernatant, the cells were diluted to 250 k / mL with fresh serum-containing PMonH plating medium. Then, 90 μL of the diluted cell suspension was plated onto a 96-well cell culture plate, with 25 k cells per well. The prepared sample working solution was added to the cell suspension to a final concentration of 0.5 nM, 1.4 nM, 4 nM, 12 nM, 37 nM, 111 nM, 333 nM, and 1,000 nM, and then placed in a 5% carbon dioxide incubator and incubated at 37 °C for 48 hours. After 48 hours, all the culture medium in the 96-well plate was aspirated, washed with 1×PBS buffer, 50 μL of the prepared Cells to CT lysis buffer (as per the manufacturer's recommendations) was added and mixed well, and after standing for 10 min, 2.5 μL of the 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 10 μL of the lysed liquid in each reaction. Gene expression quantification was measured by real-time fluorescence PCR. The TaqMan probe for monkey F11 was Mf02826986_m1, and the probe for the reference gene (monkey PPIB) was Mf02802985_m1 (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were 1 cycle of 95 °C for 20 seconds, followed by 40 cycles of 95 °C for 1 second and 60 °C for 20 seconds. The real-time fluorescence PCR instrument was QuantStudio TM6Pro Real-Time Fluorescent Quantitative PCR System (ThermoFisher). The expression of the F11 gene was calculated by 2^-ΔΔCt, and the expression of the PPIB gene was used as an internal reference. The silenced expression level of the F11 gene was calculated as a percentage compared to the cell group with only culture medium as a control. The siRNA concentration (IC50) value that reduced the F11 expression level by 50% is shown in Table 10.

[0165] Table 9

[0166] Serial number Sense strand (5’-3’) Antisense strand (5’-3’) SN-685888 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-686683 gsCfsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-686684 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgUfcuuugUfuGfcaagcsusu SN-686685 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusUf

[0167] Table 10

[0168] Serial number IC50, nM SN-685888 10.66 SN-686683 3.70 SN-686684 3.13 SN-686685 5.81

[0169] Example 6

[0170] SN-685888, which has good activity in transgenic mice, was selected for further optimization and modification (Table 11). On day 0, 3 mg / kg of the compound was subcutaneously injected into human FXI transgenic mice, and the human FXI protein in the blood was tracked and observed. The results are as Figure 2 .

[0171] Table 11

[0172] Serial number Sense strand (5’-3’) Antisense strand (5’-3’) SN-685888 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-685295 gsCfsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsAfaaUfgUfcuuugUfuGfcaagcsusUf SN-685296 gscsuugcAfaCfAfAfaGfacauuuau-TriGalNAc asUfsAfaaUfgUfcuuugUfuGfcaagcsusu SN-685297 gsCfsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsAfaaUfgUfcuuugUfuGfcaagcsusu SN-685298 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsAfaaUfgUfcuuugUfuGfcaagcsusUf SN-685299 gsCfsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaaaUfgUfcuuugUfuGfcaagcsusUf

[0173] Example 7

[0174] SN-685299, which has better in vivo effects, was selected to further verify the siRNA activity in cynomolgus monkeys. At doses of 5 mg / kg and 20 mg / kg, it was found that the FXI protein level in the serum decreased in a dose-dependent manner, with a decrease of approximately 90% and 95% ( Figure 3 ).

[0175] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A nucleic acid comprising a sense strand and an antisense strand, characterized in that: The sense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with any at least 15 consecutive nucleotides in the sequence shown in any one of SEQ ID Nos. 1 to 121; the antisense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with any at least 15 consecutive nucleotides in the nucleotide sequence shown in any one of SEQ ID Nos. 121 to 240.

2. The nucleic acid according to claim 1, wherein The antisense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with at least 15 consecutive nucleotides in any of the sequences shown in any one of SEQ ID No.125, 126, 127, 129, 165, and 166; optionally, the positive strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides compared with at least 15 consecutive nucleotides in any of the sequences shown in any one of SEQ ID No.5, 6, 7, 9, 45, and 46.

3. The nucleic acid according to claim 1, wherein The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 7 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 127 by 0, 1 or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 9 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO: 129 by 0, 1 or 2 nucleotides; or, The sense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:45 by 0, 1 or 2 nucleotides, and the antisense strand contains a nucleotide sequence that differs from the sequence shown in SEQ ID NO:165 by 0, 1 or 2 nucleotides.

4. The nucleic acid according to claim 1, wherein At least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified internucleotide; The modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-open ring nucleotide mimic, locked nucleotide, 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, reverse nucleotide, reverse 2'-O-methyl nucleotide, reverse 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide and 3'-O-methyl nucleotide; the modified nucleotide is further preferably selected from one or two of 2'-O-methyl nucleotide and 2'-fluoro nucleotide; The modified internucleotide bond is preferably selected from one or more of phosphorothioate internucleotide bonds and methylphosphonate internucleotide bonds; the modified internucleotide bond is further preferably selected from one or more of phosphorothioate monoester internucleotide bonds and phosphorothioate diester internucleotide bonds.

5. The nucleic acid according to claim 4, wherein The antisense strand has 2'-fluoro nucleotides at the 2nd, 6th, 14th and 16th nucleotide positions and at least one of the 3rd, 4th, 8th and 23rd nucleotide positions of the nucleotide sequence shown in any one of SEQ ID Nos. 121 to 240, and the other positions have 2'-O-methyl nucleotides; Preferably, the antisense strand has 2'-fluoro nucleotides at the 2nd, 6th, 8th, 14th, 16th and 23rd nucleotide positions of the nucleotide sequence as shown in any one of SEQ ID Nos. 121 to 240, and 2'-O-methyl nucleotides at other positions.

6. The nucleic acid according to claim 4 or 5, wherein The sense strand has 2'-fluoro nucleotides at the 7th, 9th, 10th and 11th nucleotide positions and at least one selected from the 2nd and 13th nucleotide positions of the nucleotide sequence shown in any one of SEQ ID Nos. 1 to 120, and the other positions have 2'-O-methyl nucleotides; Preferably, the sense strand has 2'-fluoro nucleotides at the 2nd, 7th, 9th, 10th and 11th nucleotide positions of the nucleotide sequence as shown in any one of SEQ ID No. 1 to 120, and 2'-O-methyl nucleotides at other positions.

7. The nucleic acid according to any one of claims 4 to 6, wherein The last 2 to 4 nucleotides at the 5' end and / or 3' end of the antisense strand contain a phosphorothioate internucleotide bond, and the last 2 to 4 nucleotides at the 5' end and / or 3' end of the sense strand contain a phosphorothioate internucleotide bond.

8. The nucleic acid according to claim 1, wherein The antisense strand contains a nucleotide sequence that differs from the antisense strand shown in any one of Table 2 or Table 3 by 0, 1 or 2 nucleotides; Preferably, the sense strand contains a nucleotide sequence that differs from the sense strand shown in any one of Table 2 or Table 3 by 0, 1 or 2 nucleotides; Preferably, the nucleic acid contains a duplex as shown in any one of Table 2 or Table 3.

9. The nucleic acid according to claim 8, wherein The nucleic acid contains a duplex selected from any one of SN-255886, SN-255887, SN-255888, SN-255880, SN-255826, SN-255827, SN-256683, SN-256684, SN-256685, SN-255295, SN-255296, SN-255297, SN-255298, and SN-255299.

10. A targeted drug delivery system, characterized in that: The targeted drug delivery system comprises a targeting group, a linking group, and the nucleic acid according to any one of claims 1 to 9 connected to the targeting group via the linking group.

11. The targeted drug delivery system according to claim 10, wherein: The targeted drug delivery system comprises a ligand and the nucleic acid connected to the ligand, wherein the ligand is connected to one or more of the 5' end of the antisense strand, the 3' end of the antisense strand, the 5' end of the sense strand and the 3' end of the sense strand; preferably, the ligand is a GalNAc derivative; more preferably, the ligand is one or more GalNAc derivatives connected by single-stranded, double-stranded or triple-stranded branched linkers.

12. The targeted drug delivery system according to claim 10, wherein: The structure of the targeted drug delivery system is shown below: In the formula, Nu represents the nucleic acid.

13. An isolated cell, characterized in that: The cell contains the nucleic acid according to any one of claims 1 to 9.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the nucleic acid according to any one of claims 1 to 9 or the targeted drug delivery system according to any one of claims 10 to 12 and a pharmaceutically acceptable carrier.

15. A method for inhibiting the expression of coagulation factor XI in a cell, the method comprising: The cell is contacted with the nucleic acid of any one of claims 1 to 9, the targeted drug delivery system of any one of claims 10 to 12, or the pharmaceutical composition of claim 14 to inhibit the expression of coagulation factor XI in the cell.

16. Use of the nucleic acid according to any one of claims 1 to 9, the targeted drug delivery system according to any one of claims 10 to 12, or the pharmaceutical composition according to claim 14 in any of the following aspects: 1) Treatment and / or prevention of diseases related to blood coagulation; 2) Preparation of drugs for treating and / or preventing diseases related to blood coagulation.

17. The use according to claim 16, wherein The diseases are: (i) a disease associated with an increase or elevation of factor XI; or (ii) Diseases that would benefit from reduced expression of factor XI.

18. The use according to claim 16, wherein The disease is selected from the group consisting of diseases related to abnormal coagulation, preferably at least one selected from pulmonary embolism, venous embolism, deep vein thrombosis, myocardial infarction and stroke.

19. The use according to claim 16, wherein The nucleic acid, the targeted drug delivery system or the pharmaceutical composition is administered to a subject by subcutaneous administration, intravenous administration and / or intramuscular administration.

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