Nucleic acids targeting coagulation factor XI and their applications

Abstract

This invention relates to the field of biotechnology, specifically to nucleic acids targeting coagulation factor XI and their uses. The nucleic acids of this invention can effectively reduce coagulation factor XI levels and effectively inhibit thrombus formation, thereby preventing and / or treating various diseases caused by thrombosis, such as pulmonary embolism, venous embolism, deep vein embolism, myocardial infarction, and stroke.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to nucleic acids targeting coagulation factor XI and their uses.

[0002] Cross-references to related applications This invention claims priority to Chinese Patent Application No. 202410368288.1, filed on March 28, 2024, the entire contents of which are incorporated herein by reference. Prior Technology

[0003] The coagulation mechanism is an indispensable function of human blood circulation, and normal coagulation function can prevent blood loss. The coagulation reaction generally initiates through two pathways: the extrinsic and the intrinsic pathways. The extrinsic pathway, also known as the tissue factor pathway, is initiated by tissue damage, causing tissue factor (TF or Factor III) to bind to coagulation factor VIIa, subsequently initiating factor X into Xa. The intrinsic pathway, also known as the contact initiation pathway, is initiated by surface or vascular endothelial damage, causing factor XII to convert to XIIa. Subsequently, XIIa converts factor XI to XIa, and XIa converts factor IX to IXa, thus initiating factor X into Xa. From this point, both initiation pathways enter the common pathway of the coagulation reaction: Xa binds to cofactor Va to form prothrombinase complex (also known as thrombin activation enzyme), which promotes the conversion of prothrombin (factor II) into thrombin (factor IIa). Thrombin then converts fibrinogen (factor I) into fibrin (factor Ia), and also initiates factor XIII and converts it into XIIIa, further promoting fibrin cross-linking to form a thrombus.

[0004] Abnormalities in the coagulation mechanism can lead to various diseases. For example, the deficiency of coagulation factors VIII and IX can cause hemophilia, while thrombosis and vascular embolism are the root cause of many diseases such as pulmonary embolism, deep vein thrombosis, myocardial infarction, and stroke. Clinically used antithrombotic therapies such as heparin, low molecular weight heparin, or warfarin have limitations and are prone to treatment-related bleeding. Many embolisms are believed to be caused by the initiation of the coagulation pathway after surgery. Studies on individuals with factor XI deficiency (hemophilia C) have found that their bleeding tendency is generally no different from that of normal individuals, only occurring during surgery and severe trauma. This makes factor XI a potential target for the treatment of thrombosis.

[0005] RNA interference (RNAi) refers to the highly conserved phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded small interfering RNA (siRNA) during evolution. RNAi drugs also have the advantage of longer duration of action compared to antibody drugs. Therefore, the research and development of siRNAs targeting coagulation factor XI is of great significance. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems existing in the prior art and provide a new nucleic acid targeting coagulation factor XI and its uses.

[0007] The first aspect of the present invention provides a nucleic acid comprising a sense strand and an antisense strand, wherein the sense strand contains at least 15 consecutive nucleotides, and the at least 15 consecutive nucleotides differ by no more than 3 nucleotides from any at least 15 consecutive nucleotides in any sequence shown in SEQ ID No. 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 from any at least 15 consecutive nucleotides in any sequence shown in SEQ ID No. 121 to 240.

[0008] A second aspect of the present invention provides a targeted drug delivery system comprising a target group, a linker group, and a nucleic acid as described above, which is linked to the target group via the linker group.

[0009] The present invention provides an isolated cell containing the aforementioned nucleic acid.

[0010] A fourth aspect of the present invention provides a pharmaceutical composition comprising a nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

[0011] The fifth aspect of the present invention provides a method for inhibiting the expression of coagulation factor XI in cells, the method comprising: contacting the cells with the nucleic acid, the targeted drug delivery system, or the drug composition to inhibit the expression of coagulation factor XI in the cells.

[0012] The sixth aspect of the present invention provides the use of the said nucleic acid, the said targeted drug delivery system, or the said pharmaceutical composition in any of the following aspects: 1) treating and / or preventing coagulation-related diseases; 2) preparing a medicament for treating and / or preventing coagulation-related diseases.

[0013] The nucleic acid of this invention can effectively reduce the level of coagulation factor XI and effectively inhibit the formation of thrombi, thereby preventing and / or treating various diseases caused by thrombosis, such as pulmonary embolism, venous embolism, deep vein embolism, myocardial infarction, stroke, etc. Simple Explanation of the Diagram

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort. Figure 1 shows the residual serum F11 level on day 7 after subcutaneous injection of different targeted drug delivery systems into human FXI transgenic mice in this embodiment of the invention. Figure 2 shows the results of tracking and observing the residual serum F11 level in human FXI transgenic mice after subcutaneous injection of different modified drugs in an embodiment of the present invention. Figure 3 shows the activity verification results of SN-685299 in cynomolgus monkeys in the embodiments of the present invention. Implementation

[0015] 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.

[0016] Terminology Explanation

[0017] 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.

[0018] 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 using 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 "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").

[0019] 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.

[0020] 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.

[0021] 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 allowed. For example, 100mM can fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0022] In this invention, the terms "multiple" or "various" are used, and unless otherwise specified, they refer to a quantity of 2 or more.

[0023] 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.

[0024] 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.

[0025] In this invention, "optionally," "optionally," "optionally," "optionally," "optionally," and "optional" refer to options that are optional, meaning they are selected from either "present" or "absent." If multiple "optional" or "optional" terms appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" or "optional" term is independent.

[0026] In this invention, the term "nucleic acid" refers to 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. The nucleic acid may function through RNA interference mechanisms (i.e., by interaction with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC), or through any alternative mechanism or pathway. The scope of nucleic acids disclosed herein, including both sense and antisense strands, 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.

[0027] In this invention, when it is mentioned that "the antisense strand (positive strand) contains at least 15 consecutive nucleotides, and the at least n consecutive nucleotides differ from any at least 15 consecutive nucleotides in the reference sequence (e.g., the sequence shown in SEQ ID No. X or the nucleotide sequence of positions 1-21 of the shown sequence) by no more than 3 nucleotides", the alignment involved includes alignment with at least 15 consecutive nucleotides starting at any position (e.g., positions 1, 2, 3, ..., 7, or 8) in the reference sequence. As an example, in some embodiments, the antisense strand (positive strand) contains 21 consecutive nucleotides, wherein the nucleotide sequence of positions 1-15 differs from the nucleotide sequence of positions 2-16 of the reference sequence in one, two, or three places, and the nucleotides of positions 16-20 in the antisense strand (positive strand) are all the same as or all different from the nucleotide sequence of positions 17-21 of the reference sequence; such sequences are also within the scope of this invention. In some embodiments, the antisense strand (positive strand) contains 21 consecutive nucleotides, wherein the sequence of nucleotides 1 to 15 is identical to the sequence of nucleotides 1 to 15 of the reference sequence, and nucleotides 16 to 21 of the antisense strand (positive strand) are either identical or different from the sequence of nucleotides 16 to 21 of the reference sequence; such sequences are also within the scope of this invention.

[0028] In this invention, when referring to descriptions related to sequence alignment, the term "difference" or "separation" includes one or more of the following: substitution, insertion, and deletion.

[0029] In this invention, "coagulation factor XI (FXI or F11)," also known as plasma prekallikrein, is a protein involved in the blood coagulation process. It plays an important role in the coagulation cascade, especially in the intrinsic pathway. When blood vessels are damaged, a series of enzymatic reactions are initiated, ultimately leading to fibrin formation, which helps with hemostasis and wound healing.

[0030] 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 polypeptide, protein, or protein subunit 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 nucleic acid 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Nucleic acid

[0038] This invention provides a nucleic acid (modified or unmodified) comprising a sense strand and an antisense strand. The sense strand contains at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides, and the difference between the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides and any of the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in any sequence shown in SEQ ID No. 1-121 is no more than 0, 1, 2, or 3 nucleotides. The antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, and the difference between the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides and any of ... The nucleotide sequence of any of the sequences shown in Nos. 121 to 240 differs by no more than 0, 1, 2, or 3 nucleotides from any of the following consecutive 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides.

[0039] 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 differ from any of the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in the nucleotide sequence of positions 1 to 21 of any of the sequences shown in SEQ ID No. 121 to 240 by no more than 0, 1, 2, or 3 nucleotides.

[0040] In some embodiments, the antisense strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

[0041] In some embodiments, the positive strand has 15 to 30 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30) nucleotides (bases).

[0042] In this invention, the justice chain and the antisense chain can have the same length or different lengths.

[0043] In practical implementation, those skilled in the art can combine the sequences provided in this invention by taking into account the complementarity of the sense and antisense strands, thereby obtaining combined nucleic acids (such as siRNA).

[0044] In a preferred embodiment of the present invention, as shown in Table 1, the nucleic acid is selected from at least one of the following: siRNA-1 with the sense strand sequence SEQ ID No. 1 and the antisense strand sequence SEQ ID No. 121; siRNA-2 with the sense strand sequence SEQ ID No. 2 and the antisense strand sequence SEQ ID No. 122; siRNA-3 with the sense strand sequence SEQ ID No. 3 and the antisense strand sequence SEQ ID No. 123; siRNA-4 with the sense strand sequence SEQ ID No. 4 and the antisense strand sequence SEQ ID No. 124; siRNA-5 with the sense strand sequence SEQ ID No. 5 and the antisense strand sequence SEQ ID No. 125; siRNA-118; siRNA-119; and siRNA-120.

[0045] In some preferred embodiments, the antisense strand contains at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides, and the difference between the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides and any of the at least 15, 16, 17, 18, 19, 20, 21, 22, or 23 consecutive nucleotides in any of the sequences shown in SEQ ID No. 125, 126, 127, 129, 165, and 166 is no more than 3 nucleotides; the sense strand contains a nucleotide sequence that is at least partially complementary (e.g., partially complementary, substantially complementary, or completely complementary) to the antisense strand. When the antisense strand has the above sequence, the double-stranded RNA exhibits a significantly better inhibitory effect on coagulation factor XI.

[0046] In some preferred embodiments, the positive 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 the at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides in any of the sequences shown in SEQ ID No. 5, 6, 7, 9, 45, 46.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] All nucleotide groups in the above nucleic acids may be unmodified or contain at least one modified nucleotide group, and the modification may be on nucleotides at any position.

[0051] In some implementations, the justice chain and the antisense chain may be partially complementary, substantially complementary, or completely complementary to each other.

[0052] In some embodiments, when the sequence identity of the sense or antisense strand of the nucleic acid with the corresponding sequence mentioned in this invention is less than 100% or differs by more than one nucleotide, it still has an inhibitory effect on coagulation factor XI 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 any of the sequences shown in SEQ ID No. 121-240) can be replaced with UU, AA, CU, UC, AG, CC, GG, or UG, or any combination of two nucleic acids. Such nucleic acid sequences are also within the scope of protection of this invention.

[0053] In some preferred embodiments, the nucleic acid has an inhibitory efficiency of not less than 50% against coagulation factor XI (e.g., 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%).

[0054] Table 1 Targeted gene loci Chain of Justice (5'-3') SEQ ID NO. Antisense chain (5'-3') SEQ ID NO. 385 GCAGCGAUUUCUGGGUAUUCU 1 AGAAUACCCAGAAAUCGCUGCUU 121 387 AGCGAUUUCUGGGUAUUCUUU 2 AAAGAAUACCCAGAAAUCGCUGC 122 389 CGAUUUCUGGGUAUUCUUUCA 3 UGAAAGAAUACCCAGAAAUCGCU 123 411 GCAAUGCUCACACCAAAUAAG 4 CUUAUUUGGUGUGAGCAUUGCUU 124 430 AGCGCUUGCAACAAAGACAUU 5 AAUGUCUUUGUUGCAAGCGCUUU 125 431 GCGCUUGCAACAAAGACAUUU 6 AAAUGUCUUUGUUGCAAGCGCUU 126 433 GCUUGCAACAAAGACAUUUAU 7 AUAAAUGUCUUUGUACAAGCUU 127 462 AGACAUGAAGGGCAUAAAACUA 8 UAGUUAUGCCCCUUCAUGUCUUU 128 463 GACAUGAAGGCAUAAACUAU 9 AUAGUUUAUGCCCUUCAUGUCUU 129 468 GAAGGGCAUAACUAUAACAG 10 CUGUUAUAGUUUAUGCCCUUCUU 130 484 AACAGCUCAGUUGCCAAGU 11 ACUCUUGGCAACUGAGCUGUUUUU 131 487 AGCUCAGUUGCCAAGAGUGCU 12 AGCACUCUUGGCAACUGACGCU 132 493 GUUGCAAGAGUGCUCAAGAA 13 UUCUUGAGCACUCUUGGCAACUG 133 494 UUGCCAAAGUGCUAAAA 14 UUUCUUGAGCACUCUUGGCAACU 134 503 GUGCUCAGAAUGCCAAGAAA 15 UUUCUUGGCAUUCUUGAGCACUC 135 505 GCUCAAGAAUGCCAAGAAAGA 16 UCUUUCUUGGCAUUCUUGAGCAC 136 556 ACGUACGCCACAAGGCAGUUU 17 AAACUGCCUUGUGGCGUACGUUU 137 580 AGCCUGGAGCAUCGUAACAUU 18 AAUGUUACGAUGCUCCAGGCUUU 138 622 ACAGGGACACCAACCAGAAUA 19 UAUUCUGGUUGGUCUUUU 139 678 GAAAUCCUGUGCACAUUUCUAA 20 UUAGAAAAGUGCACAGGAUUUCU 140 679 AAAUCCUGUGCACUUUCUAAU 21 AUUAGAAAGUGCACAGGAUUUUU 141 681 AUCCUGUGCACUUUCUAAUCU 22 AGAUUAGAAAGUGCACAGGAUUU 142 690 ACUUUCUAAUCUGGCUUGUAU 23 AUACAAGCCAGAUUAGAAAGUGC 143 741 AGACAGCAACAUCGACAGUGU 24 ACACUGUCGAUGUUGCUUUU 144 787 GGCCGAAUCUGCCACUCAUCAU 25 AUGAUGAGUGCAGAUUCGGCCUU 145 845 GGCCCAAAGAAUCUAAAGA 26 UUCUUUGAGAUUCUUUGGGCCUU 146 846 GCCCAAAGAAUCUCAAAGAAA 27 UUUCUUGAGAUUCUUGGGCUU 147 849 CAAGAAUCUAAAGAAUCU 28 AGAUUUCUUGAGAUUUCUUGU 148 853 GAAUCUCAAAGAAUCUUGU 29 ACAAAGAUUUCUUGAGAUUCU 149 868 CUUUGUCUCCUUAAAACAUCU 30 AGAUGUUUUAAAGGAGACAAAGUU 150 889 GAGAGUGGAUUGCCCAGUACA 31 UGUACUGGGCAAUCCACUCUU 151 981 CCAUUCUUCAUUUUACCAUGA 32 UCAUGGUAAAUGAAGAAUGGUU 152 1014 GGGAGAAGAACUGGAUAUUGU 33 ACAAUAUCCAGUUCUUCCCCUU 153 1015 GGAGAAGAACUGGAUAUUGUU 34 AACAAUAUCCAGUUCUUCCCUU 154 1027 GAUAUUGUUGCUGCAAAAAGU 35 ACUUUUUGCAGCAACAAUAUCCA 155 1052 AGGCCUGCCAGAAACUGUGCA 36 UGCACAGUUUCUGGCAUUCCUCG 156 1062 GAAACGUGCACCAAUGCCGU 37 ACGGCAUUGGUGCACAGAUUUCUG 157 1171 GGAUCUCCAACUAAAUACUU 38 AAGUAUUUUAUGUUGGAGAUCCUU 158 1199 GAGGAGGCAUCUCUGGAUACA 39 UGUAUCCAGAGAUGCCUCCUCU 159 1232 GUAAAAUGGAUAAUGAGUGUA 40 UACACUCAUUAUCCAUUUUACAC 160 1276 GUUGGAGGAACUGCGUCUGUU 41 AACAGACGCAGUUCCUCCCAACUU 161 1372 AUUGGAAACCAGUGGAAUAUUA 42 UAAUAUCCACUGGUUUCCAAUUU 162 1375 GGAAACCAGUGGAAUUAACA 43 UGUUAAUAUCCACUGGUUUCCAA 163 1415 GGGUAGAGUCACCUAAGAUUU 44 AAAUCUUAGGUGACUCUACCCCUU 164 1451 GCAUUUUAAAUCAUCUGAA 45 UUUCAGAUUGAUUUAAAAUGCUU 165 1452 CAUUUUAAAUCAUCUGAAAU 46 AUUUCAGAUUGAUUUAAAAUGUU 166 1503 AGAAAUAAUAAUCCAUGAUCA 47 UGAUCAUGGAUUAUUAUUUCUUU 167 1504 GAAAUAAUAAUCCAUGAUCAG 48 CUGAUCAUGGAUUAUUAUUUCUU 168 1506 AAUAAUAAUCCAUGAUCAGUA 49 UACUGAUCAUGGAUUAUUAUUUC 169 1533 GGCAGAAAGCGGGUAUGAUAU 50 AUAUCAUACCCGCUUUCUGCCAU 170 1534 GCAGAAAGCGGGUAUGAUAUU 51 AAUAUCAUACCCGCUUUCUGCCA 171 1623 AGGAGAUAGAAAUGUAAUAUA 52 UAUAUUACAUUUCUAUCUCCUUU 172 1675 AGAAAACUAAGAGACAAAAUA 53 UAUUUUGUCUCUUAGUUUUCUGU 173 1702 ACUCUCCAGAAAGCCAAGAUA 54 UAUCUUGGCUUUCUGGAGAGUAU 174 1710 GAAAGCCAAGAUACCCUUAGU 55 ACUAAGGGUAUCUUGGCUUUCUG 175 1741 GAGUGCCAGAAGAGAUACAG 56 UCUGUAUCUCUUCUGGCACUCUU 176 1745 GCCAGAAGAGAUACAGAGAC 57 GUCCUCUGUAUCUCUGGCAC 177 1748 AGAAGAGAUACAGAGGACAUA 58 UAUGUCCUCUCUGUAUCUCUGG 178 1749 GAAGAGAUACAGAGCACUAA 59 UUAUGUCCUCUGUAUCUCUUCUG 179 1750 AAGAGAUACAGGACAUAAA 60 UUUAUUGUCCUCUGUAUCUCUUCU 180 1752 GAGAUACAGAGGACAUAAAAU 61 AUUUUAUUGUCCUCUGUAUCUCUU 181 1754 GAUACAGAGGACAUAAAAAA 62 UUAUUUUAUUGUCCUCUGUAUCUG 182 1762 GGACAUAAAAUAAACCCUAAG 63 CUUAUGGGUUAUUUAUUGUCCUC 183 1763 GACAUAAAAUAACCCAUAAGA 64 UCUUAUGGGUUAUUUAUUGUCUG 184 1969 GAGAAAACUCAAGCAGUGUGA 65 UCACACUGCUUGAGUUUUCUCUU 185 1971 GAAAACUCAAGCAGUGUGAAU 66 AUUCACACUGCUUGAGUUUUCUC 186 2109 GUAAGAAAAUGCUAGAAGAAA 67 UUUCUUCUAGCAUUUUCUUACUU 187 2113 GAAAAUGCUAGAAGAAAACAA 68 UUGUUUUCUUCUAGCAUUUUCUU 188 2122 AGAAGAAAACAAACUGUCACA 69 UGUGACAGUUUGUUUUCUUUCUAG 189 2131 CAAACUGUCACAAGUUGUUAU 70 AUAACAUUGUGAGACAGUUGUU 190 2165 GUUCUAUGAUCGUUGUAGUUU 71 AAACUACAACGAUCAUAGACUU 191 2182 GUUUGUUGAGCAUCAGUCU 72 AGACUGAAUGCUCAAACAAACUU 192 2227 GGAGUCCAAGAAUUACCAUAA 73 UUAUGGUAAUUCUUGGACUCCUU 193 2236 GAAUUACCAUAAGGCAAUAUU 74 AAUAUUGCCUUAUGGUAAUUCUU 194 2249 GCAAAUUUCUGAAGAUUACU 75 AQUAAUCUUCAGAAAUAUUGCUU 195 2275 GGCAGAUAUAGCAGAAAAUAA 76 UUAUUUUCUGCUAUAUCUGCCUU 196 2319 GGGCAGAAGACUGGUAAAAGA 77 UCUUUUACCAGUUCUUCUGCCUU 197 2320 GCAGAAGAACUGGUAAAGAA 78 UUCUUUUACCAGUUCUUCUGCUU 198 2331 GGUAAAAGAAGCCACCAUAAA 79 UUUAUGGUGGCUUCUUUUUACCUU 199 2359 GUUCGAUGAAAGAUGAAACU 80 AGUUUUCAUCUUCAUCGAACAA 200 2373 GAAAACUGGAAGGAAGGAAA 81 UUCUCCUUUCUUCCAGUUUUCUU 201 2381 GAGAAAGGAGAAAAAAGA 82 UGUCUUUGUUCUCCUUUCUUCUU 202 2383 AGAAAGGACAAAGAGAGU 83 ACUGUCUUUGUUCUCCUUUCUUC 203 2420 GAAUCUACACUCUGCCUAUGU 84 ACAAUGGCAGAGUGUAAGAUCUU 204 2434 CCUAUGUAACACAUUUCUUU 85 AAAGAAAAUGUGUUCACAUAGGCAA 205 2435 CUAUGUGAACACAUUUUUUU 86 AAAAGAAAUGUGUUCACAUAGUU 206 2475 GCAUUUAAUGGCAGUUUUCA 87 UGAAAAUCUGCCCAUUAAUUGCAA 207 2505 GGAAUUCUUGUCAUUUCCAUU 88 AAUGGAAAUGACAAGAAUUCCUU 208 2506 GAAUUCUUGUCAUUUCCAUUU 89 AAAUGGAAAUGACAAGAAUUCUU 209 2551 GUUCGAGUAGACAGAGCUAA 90 UUAGCUCGUCUACUGUACUCGAACG 210 2561 ACACGAGCUAAGAGUGAUGU 91 ACAUUCACUCUUAGCUGUGUUU 211 2563 ACGAGCUAAAGAGUGAAUGUGA 92 UCACAUUCACUCUUAGCUGUGU 212 2565 GACUAAGAGUGAAUGUGAAG 93 CUUCACAUUCACUCUUAGCUCUU 213 2566 AGCUAAGAGUGAAUGUGAAGA 94 UCUUCACAUUCACUCUUAGCUCG 214 2567 GCUAAGAGUGAAUGUAAGAU 95 AUCUUCACAUUCACUCUUAGCUC 215 2571 AGAGUGAAUGUGAAGAUAACA 96 UGUUAUCUUCACAUUCACUCUUA 216 2573 AGUGAAUGUGAAGAUAACAGA 97 UCUGUUAUCUUCACAUUCACUCU 217 2576 GAAUGUGAAGAUAACAGAAUU 98 AAUUCUGUUAUCUUCACAUUCAC 218 2609 AGGAUUACAAGCAGCAAUUUA 99 UAAAUUGCUGCUUGUAUCCUUU 219 2622 GCAAUUUACCUGGAAGUGUA 100 UAUCACUUCCAGGUAAAUUGCUU 220 2682 GAUUUGUGAUGGAUUGUAUAU 101 AUAUACAAUCCAUCACAAAUCUU 221 2687 GUGAUGGAUUGUAUAUUUAUU 102 AAUAAAUUACAAUCCAUCACAA 222 2689 GAUGGAUUGUAUAUUUAUUUA 103 UAAAUAAAUAUACAAUCCAUCAC 223 2750 GCUCAAACCUCCCUAAGACAA 104 UUGUCUAGGGAGGGUUGAGCUU 224 2766 GACAAGCUGCUGCUGUGACUA 105 UAGUCACAGCAGCAGCUUGUCUA 225 2799 GAGCUAGAUCGUAUAUUUAUU 106 AAUAAAUAUACGAUCUAGCUCUU 226 2841 GCAUCCAUACUACAGAGAAAAA 107 UUUUCUCUGUAGUAUGGAUGCAG 227 2874 GCAAAUGGAUAGUAGUUACAGUAA 108 UUACUGUAACUAUCCAUUUGCUU 228 2875 CAAUGGAUAGUAGUUACAGUAA 109 UUUACUGUAACUAUCCAUUUGUU 229 2939 CUGUUUCGUGCAAAUAUUUAU 110 AUAAAAUAUUUGCACGAAACAGUU 230 2941 GUUUCGUGCAAAUAUUUAUCU 111 AGAUAAAUAUUUGCACGAAACAG 231 2946 GUGCAAAUAUUUAUCUCAUUA 112 UAUUGAGAUAAAUAUUUGCACGA 232 2948 GCAAAUAUUUAUUCUCAUUAUU 113 AAUAAUGAGAUAAAAUAUUUGCAC 233 2974 GAUCUAGUUCAAUAACCUAGA 114 UCUAGGUUAUUGACUAGAUCUU 234 2980 GUUCAAUAACCUAGAAUUUGA 115 UCAAAUUCUAGGUUAUUGACUU 235 2983 CAUAACCUAGAUUUGAAUU 116 AAUUCAAAUUCUAGGUUAUUGUU 236 3007 ACCACAUAGCUUUCAAUCUGU 117 ACAGAUUGAAAGCUAUUGGUUU 237 3020 CAAUCUGUGCCAACAUUA 118 UAUAGUUGUUGGCACAGAUUGUU 238 3028 GCCAACAACUAUACAAUUCAU 119 AUGAAUUGUAUAGUUGUUGGCUU 239 3030 CAACAACUAUACAAUUCAUCA 120 UGAUGAAUUGUAUAGUUGUUGUU 240

[0055] In Table 1, column 1 indicates the position of the first base of the target gene in the coding sequence of human coagulation factor XI, and so on; the numbers in columns 3 and 5 represent sequence numbers, for example, "1" represents SEQ ID No. 1. The reference sequence of the target gene is the coding sequence of human coagulation factor XI, NM_000128.4.

[0056] The advantages of the above-mentioned technical solutions regarding bare sequences (i.e., unmodified sequences) mentioned in this invention do not depend on the choice of modification method or target vector. The following section elaborates on applicable modification schemes and further preferred modification schemes.

[0057] 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 inhibitory efficiency of not less than 50% against coagulation factor XI (e.g., 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] In some embodiments, the modified nucleotide group is a nucleotide group whose phosphate group and / or ribose group are modified. The modified site can be at least position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of the nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of the nucleotides in the sense strand and / or antisense strand.

[0059] In some embodiments, modification of the phosphate group refers to modifying the oxygen in the phosphate group, including phosphate thioate modification and boranophosphate modification. The oxygen in the phosphate group is replaced by sulfur, borane, amino, alkyl, or alkoxy groups, as shown in the following formulas. These modifications can stabilize the structure of nucleic acids and maintain high specificity and high affinity of base pairing.

[0060] In the above structural formulas, BASE represents a 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 structures, 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, acetoxybenzyl, acetoxyethyl.

[0061] In some embodiments, modification of the ribose group refers to the modification of the 2′-hydroxyl group (2′-OH) in the ribose group. Introducing certain substituents, such as methoxy or fluorine, at the 2′-hydroxyl position of the ribose group makes the nucleic acid less susceptible to cleavage by ribonucleases, thereby increasing the stability of the nucleic acid and giving it stronger resistance to nuclease hydrolysis. Modification of the 2′-hydroxyl group in the pentose of nucleotides includes 2′-fluoromodification (e.g., 2′-arabino-fluoro modification), 2′-methoxymodification (2′-OME), 2′-methoxyethylmodification (2′-MOE), 2′-2,4-dinitrophenol modification (2′-DNP modification), 2′,4′-constrained ethylmodification, 2′-aminomodification (2′-Amino modification), 2′-deoxymodification (2′-Deoxy modification), BNA, acyclic nucleic acid modification, misaligned nucleic acid modification, L-type nucleic acid modification, etc. BNA (inner-ring bridging nucleotide) refers to a restricted or inaccessible nucleotide. BNAs can contain bridging structures with "fixed" C 3'-endoglycosylation rings, including five-, six-, or seven-membered rings. This bridge is typically incorporated into the 2'-, 4'-positions of the ribosome to provide 2',4'-BNA nucleotides, such as locked-ethyl (LNA), ring-locked-ethyl (ENA), and ethyl-locked nucleic acid (cET BNA). Acyclic nucleic acids are nucleotides formed by opening the sugar ring of the nucleotide, such as unlocked nucleic acid (UNA) and glycerol nucleic acid (GNA). Misalignment refers to the replacement of the 3',5'-phosphate bond with a 2',5'-phosphate bond. L-type nucleic acid modification refers to the replacement of a naturally occurring D-type nucleic acid with its mirror-image L-type nucleic acid.

[0062] In this context, BASE represents the bases A, U, C, G, or T. R can be the same or different in the above structures, for example: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, acetyl, etc.

[0063] In some embodiments, according to a particularly preferred embodiment of the invention, wherein the nucleotide group containing a uracil base or a cytosine base in the justice strand of the RNAi reagent is the nucleotide group to which the ribose group is modified, i.e., the nucleotide group containing the uracil base or cytosine base in the justice strand of the RNAi reagent is substituted with a 2'-riboOH group in the nucleotide group containing the uracil base or cytosine base. More preferably, the 3' end of both the justice strand and the antisense strand of the RNAi reagent may be linked with a dTdT; The RNAi reagents with the above modifications exhibited even better in vivo inhibitory effects, and the above modifications were able to further reduce the immunogenicity of the RNAi reagents of the invention in vivo.

[0064] The RNAi reagents of the invention may also include modifications linking a single phosphate nucleoside at the 5' end of the antisense strand. Since siRNA guide-strand-terminated 5'-monophosphate is important for RISC recognition. Among them, the phosphorylation of the 5'-hydroxy group plays a certain role in whether siRNA can be efficiently loaded onto Ago2 inside the cell. The monophosphate at the 5′ end of the guide strand in siRNA interacts with Argonaute-2 (Ago2) with an H-bond, thereby ensuring accurate localization and precise cleavage of the target of the mRNA. The commonly used derivatives of 5′-monophosphate nucleosides are as follows, and such derivatives of phosphonucleosides have been confirmed to have some stability in biological metabolic mediators and play a role in promoting the loading of siRNA guide strands into Ago2 inside the cell (Nucleic Acids Research, 2015, 43, 2993–3011). RNAi reagents according to the invention, wherein, preferably trans-vinyl phosphate (VP) is preferred, may also include derivatives of monophosphate nucleosides other than those described above.

[0065] In the above structures, BASE represents the base A, U, C, G, or T. R can be the same or different in the above structures, such as: hydrogen (H), fluorine (F), methoxy (OME) or methoxyethyl (MOE), hydroxyl, allyl, ethylamino, propargyl, cyanoethyl, amino, acetyl, etc.

[0066] In this invention, , and The meaning is consistent, referring to a chemical element X being attached to any one or more groups.

[0067] In some embodiments, at least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified interphase bond.

[0068] In some embodiments, the modified nucleotide is preferably selected from one or more of 2'-O-methyl nucleotides, 2'-fluoronucleotides, 2'-deoxynucleotides, 2',3'-open-ring nucleotide mimics, locked nucleotides, 2'-F-arabinonucleotides, 2'-methoxyethyl nucleotides, debased nucleotides, ribitols, reverse nucleotides, reverse 2'-O-methyl nucleotides, reverse 2'-deoxynucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholinonucleotides, nucleotides containing vinylphosphonates, nucleotides containing cyclopropylphosphonates, and 3'-O-methyl nucleotides; the modified nucleotide is further preferably selected from one or both of 2'-O-methyl nucleotides and 2'-fluoronucleotides.

[0069] In some embodiments, the modified inter-linked bonds are preferably one or more selected from phosphate thioester nucleotide inter-linked bonds and methylphosphonate nucleotide inter-linked bonds. In some embodiments, the modified inter-linked bonds are further preferably one or more selected from phosphate thioester monoester nucleotide inter-linked bonds and phosphate thioester diester nucleotide inter-linked bonds.

[0070] In some preferred embodiments, the antisense strand is a 2'-fluoronucleotide at nucleotide positions 2, 6, 14, and 16, and at least one of nucleotide positions selected from 3, 4, 8, and 23 (e.g., position 1, 2, 3, or 4), and the other positions are 2'-O-methylnucleotides. Compared with known modification methods, the above-described antisense strand modification scheme further enhances the inhibitory effect of the nucleic acid on coagulation factor XI.

[0071] In some further preferred embodiments, the antisense strand has 2'-fluoronucleotides at positions 2, 6, 8, 14, 16 and 23 of the nucleotide sequence shown in any of SEQ ID No. 121-240, and 2'-O-methylnucleotides at other positions.

[0072] In some preferred embodiments, the positive strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11 of the nucleotide sequences shown in any of SEQ ID Nos. 1-120, and at least one (e.g., one or two) selected from positions 2 and 13, with the remaining positions containing 2'-O-methylnucleotides. Compared to known modification methods, the above-described positive strand modification scheme further enhances the inhibitory effect of the nucleic acid on coagulation factor XI.

[0073] In some further preferred embodiments, the positive strand has 2'-fluoronucleotides at positions 2, 7, 9, 10 and 11 of the nucleotide sequence shown in any of SEQ ID No. 1 to 120, and 2'-O-methylnucleotides at the other positions.

[0074] In some embodiments, the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' and / or 3' ends of the antisense strand contain phosphate thioester nucleotide bonds, and the last 2 to 4 (e.g., 2, 3, or 4) nucleotides at the 5' and / or 3' ends of the sense strand contain phosphate thioester nucleotide bonds.

[0075] In some specific embodiments, the last three nucleotides at the 5' and 3' ends of the antisense strand contain a phosphate thioester nucleotide bond, and the last three nucleotides at the 5' end of the sense strand contain a phosphate thioester nucleotide bond.

[0076] In some embodiments, the antisense strand contains a nucleotide sequence that differs from any of the antisense strands shown in Table 2 or Table 3 by 0, 1, or 2 nucleotides.

[0077] In some embodiments, the positive strand contains a nucleotide sequence that differs from any of the positive strands shown in Table 2 or Table 3 by 0, 1, or 2 nucleotides.

[0078] In some embodiments, the nucleic acid contains a double strand as shown in either Table 2 or Table 3.

[0079] In some preferred embodiments, the nucleic acid contains a double strand 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.

[0080] Table 2 serial number Chain of Justice (5'-3') Antisense chain (5'-3') SN-255872 gscsagcgAfuUfUfCfuggguauucu asGfsaAfuAfcccagaaAfuCfgcugcsusu SN-255883 asgscgauUfuCfUfGfgguauucuuu asAfsaGfaAfuacccagAfaAfucgcusgsc SN-255884 csgsauuuCfuGfGfGfuauucuuuca usGfsaAfaGfaauacccAfgAfaaucgscsu SN-255885 gscsaaugCfuCfAfCfaccaaauaag csUfsuAfuUfuggugugAfgCfauugcsusu SN-255886 asgscgcuUfgCfAfAfcaaagacauu asAfsuGfuCfuuuguugCfaAfgcgcususu SN-255887 gscsgcuuGfcAfAfCfaaagacauuu asAfsaUfgUfcuuuguuGfcAfagcgcsusu SN-255888 gscsuugcAfaCfAfAfagacauuuau asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-255889 asgsacauGfaAfGfGfgcauaaacua usAfsgUfuUfaugcccuUfcAfugucususu SN-255880 gsascaugAfaGfGfGfcauaaacuau asUfsaGfuUfuaugcccUfuCfaugucsusu SN-255881 gsasagggCfaUfAfAfacuauaacag csUfsgUfuAfuaguuuaUfgCfccuucsusu SN-255882 asascagcUfcAfGfUfugccaagagu asCfsuCfuUfggcaacuGfaGfcuguususu SN-255893 asgscucaGfuUfGfCfcaagagugcu asGfscAfcUfcuuggcaAfcUfgagcusgsu SN-255894 gsusugccAfaGfAfGfugcucaagaa usUfscUfuGfagcacucUfuGfgcaacsusg SN-255895 ususgccaAfgAfGfUfgcucaagaaa usUfsuCfuUfgagcacuCfuUfggcaascsu SN-255896 gsusgcucAfaGfAfAfugccaagaaa usUfsuCfuUfggcauucUfuGfagcacsusc SN-255897 gscsucaaGfaAfUfGfccaagaaaga usCfsuUfuCfuuggcauUfcUfugagcsasc SN-255898 ascsguacGfcCfAfCfaaggcaguuu asAfsaCfuGfccuugugGfcGfuacgususu SN-255899 asgsccugGfaGfCfAfucguaacauu asAfsuGfuUfacgaugcUfcCfaggcususu SN-255890 ascsagggAfcAfCfCfaaccagaaua usAfsuUfcUfgguugguGfuCfccugususu SN-255891 gsasaaucCfuGfUfGfcacuuucuaa usUfsaGfaAfagugcacAfgGfauuucsusu SN-255892 asasauccUfgUfGfCfacuuucuaau asUfsuAfgAfaagugcaCfaGfgauuususu SN-255803 asusccugUfgCfAfCfuuucuaaucu asGfsaUfuAfgaaagugCfaCfaggaususu SN-255804 ascsuuucUfaAfUfCfuggcuuguau asUfsaCfaAfgccagauUfaGfaaagusgsc SN-255805 asgsacagCfaAfCfAfucgacagugu asCfsaCfuGfucgauguUfgCfugucususu SN-255806 gsgsccgaAfuCfUfGfcacucaucau asUfsgAfuGfagugcagAfuUfcggccsusu SN-255807 gsgscccaAfaGfAfAfucucaaagaa usUfscUfuUfgagauucUfuUfgggccsusu SN-255808 gscsccaaAfgAfAfUfcucaaagaaa usUfsuCfuUfugagauuCfuUfugggcsusu SN-255809 csasaagaAfuCfUfCfaaagaaaucu asGfsaUfuUfcuuugagAfuUfcuuugsusu SN-255800 gsasaucuCfaAfAfGfaaaucuuugu asCfsaAfaGfauuucuuUfgAfgauucsusu SN-255801 csusuuguCfuCfCfUfuaaaacaucu asGfsaUfgUfuuuaaggAfgAfcaaagsusu SN-255802 gsasgaguGfgAfUfUfgcccaguaca usGfsuAfcUfgggcaauCfcAfcucucsusu SN-255813 cscsauucUfuCfAfUfuuuaccauga usCfsaUfgGfuaaaaugAfaGfaauggsusu SN-255814 gsgsgagaAfgAfAfCfuggauauugu asCfsaAfuAfuccaguuCfuUfcucccsusu SN-255815 gsgsagaaGfaAfCfUfggauauuguu asAfscAfaUfauccaguUfcUfucuccsusu SN-255816 gsasuauuGfuUfGfCfugcaaaaagu asCfsuUfuUfugcagcaAfcAfauaucscsa SN-255817 asgsgccuGfcCfAfGfaaacugugca usGfscAfcAfguuucugGfcAfuuccuscsg SN-255818 gsasaacuGfuGfCfAfccaaugccgu asCfsgGfcAfuuggugcAfcAfguuucsusg SN-255819 gsgsaucuCfcAfAfCfuaaaauacuu asAfsgUfaUfuuuaguuGfgAfgauccsusu SN-255810 gsasggagGfcAfUfCfucuggauaca usGfsuAfuCfcagagauGfcCfuccucsusu SN-255811 gsusaaaaUfgGfAfUfaaugagugua usAfscAfcUfcauuaucCfaUfuuuacsasc SN-255812 gsusuggaGfgAfAfCfugcgucuguu asAfscAfgAfcgcaguuCfcUfccaacsusu SN-255823 asusuggaAfaCfCfAfguggauauua usAfsaUfaUfccacuggUfuUfccaaususu SN-255824 gsgsaaacCfaGfUfGfgauauuaaca usGfsuUfaAfuauccacUfgGfuuuccsasa SN-255825 gsgsguagAfgUfCfAfccuaagauuu asAfsaUfcUfuaggugaCfuCfuacccsusu SN-255826 gscsauuuUfaAfAfUfcaaucugaaa usUfsuCfaGfauugauuUfaAfaaugcsusu SN-255827 csasuuuuAfaAfUfCfaaucugaaau asUfsuUfcAfgauugauUfuAfaaaugsusu SN-255828 asgsaaauAfaUfAfAfuccaugauca usGfsaUfcAfuggauuaUfuAfuuucususu SN-255829 gsasaauaAfuAfAfUfccaugaucag csUfsgAfuCfauggauuAfuUfauuucsusu SN-255820 asasuaauAfaUfCfCfaugaucagua usAfscUfgAfucauggaUfuAfuuauususc SN-255821 gsgscagaAfaGfCfGfgguaugauau asUfsaUfcAfuacccgcUfuUfcugccsasu SN-255822 gscsagaaAfgCfGfGfguaugauauu asAfsuAfuCfauacccgCfuUfucugcscsa SN-255933 asgsgagaUfaGfAfAfauguaauaua usAfsuAfuUfacauuucUfaUfcuccususu SN-255934 asgsaaaaCfuAfAfGfagacaaaaua usAfsuUfuUfgucucuuAfgUfuuucusgsu SN-255935 ascsucucCfaGfAfAfagccaagaua usAfsuCfuUfggcuuucUfgGfagagusasu SN-255936 gsasaagcCfaAfGfAfuacccuuagu asCfsuAfaGfgguaucuUfgGfcuuucsusg SN-255937 gsasgugcCfaGfAfAfgagauacaga usCfsuGfuAfucucuucUfgGfcacucsusu SN-255938 gscscagaAfgAfGfAfuacagaggac gsUfscCfuCfuguaucuCfuUfcuggcsasc SN-255939 asgsaagaGfaUfAfCfagaggacaua usAfsuGfuCfcucuguaUfcUfcuucusgsg SN-255930 gsasagagAfuAfCfAfgaggacauaa usUfsaUfgUfccucuguAfuCfucuucsusg SN-255931 asasgagaUfaCfAfGfaggacauaaa usUfsuAfuGfuccucugUfaUfcucuuscsu SN-255932 gsasgauaCfaGfAfGfgacauaaaau asUfsuUfuAfuguccucUfgUfaucucsusu SN-255943 gsasuacaGfaGfGfAfcauaaaauaa usUfsaUfuUfuauguccUfcUfguaucsusg SN-255944 gsgsacauAfaAfAfUfaacccauaag csUfsuAfuGfgguuauuUfuAfuguccsusc SN-255945 gsascauaAfaAfUfAfacccauaaga usCfsuUfaUfggguuauUfuUfaugucsusg SN-255946 gsasgaaaAfcUfCfAfagcaguguga usCfsaCfaCfugcuugaGfuUfuucucsusu SN-255947 gsasaaacUfcAfAfGfcagugugaau asUfsuCfaCfacugcuuGfaGfuuuucsusc SN-255948 gsusaagaAfaAfUfGfcuagaagaaa usUfsuCfuUfcuagcauUfuUfcuuacsusu SN-255949 gsasaaauGfcUfAfGfaagaaaacaa usUfsgUfuUfucuucuaGfcAfuuuucsusu SN-255940 asgsaagaAfaAfCfAfaacugucaca usGfsuGfaCfaguuuguUfuUfcuucusasg SN-255941 csasaacuGfuCfAfCfaaguuguuau asUfsaAfcAfacuugugAfcAfguuugsusu SN-255942 gsusucuaUfgAfUfCfguuguaguuu asAfsaCfuAfcaacgauCfaUfagaacsusu SN-255953 gsusuuguUfuGfAfGfcauucagucu asGfsaCfuGfaaugcucAfaAfcaaacsusu SN-255954 gsgsagucCfaAfGfAfauuaccauaa usUfsaUfgGfuaauucuUfgGfacuccsusu SN-255955 gsasauuaCfcAfUfAfaggcaauauu asAfsuAfuUfgccuuauGfgUfaauucsusu SN-255956 gscsaauaUfuUfCfUfgaagauuacu asGfsuAfaUfcuucagaAfaUfauugcsusu SN-255957 gsgscagaUfaUfAfGfcagaaaauaa usUfsaUfuUfucugcuaUfaUfcugccsusu SN-255958 gsgscagaAfgAfAfCfugguaaaaga usCfsuUfuUfaccaguuCfuUfcugccsusu SN-255959 gscsagaaGfaAfCfUfgguaaaagaa usUfscUfuUfuaccaguUfcUfucugcsusu SN-255950 gsgsuaaaAfgAfAfGfccaccauaaa usUfsuAfuGfguggcuuCfuUfuuaccsusu SN-255951 gsusucgaUfgAfAfAfgaugaaaacu asGfsuUfuUfcaucuuuCfaUfcgaacsasa SN-255952 gsasaaacUfgGfAfAfgaaaggagaa usUfscUfcCfuuucuucCfaGfuuuucsusu SN-255963 gsasagaaAfgGfAfGfaacaaagaca usGfsuCfuUfuguucucCfuUfucuucsusu SN-255964 asgsaaagGfaGfAfAfcaaagacagu asCfsuGfuCfuuuguucUfcCfuuucususc SN-255965 gsasaucuAfcAfCfUfcugccuaugu asCfsaUfaGfgcagaguGfuAfgauucsusu SN-255966 cscsuaugUfgAfAfCfacauuucuuu asAfsaGfaAfauguguuCfaCfauaggscsa SN-255967 csusauguGfaAfCfAfcauuucuuuu asAfsaAfgAfaauguguUfcAfcauagsusu SN-255968 gscsauuuAfaUfGfGfcagauuuuca usGfsaAfaAfucugccaUfuAfaaugcsasa SN-255969 gsgsaauuCfuUfGfUfcauuuccauu asAfsuGfgAfaaugacaAfgAfauuccsusu SN-255960 gsasauucUfuGfUfCfauuuccauuu asAfsaUfgGfaaaugacAfaGfaauucsusu SN-255961 gsusucgaGfuAfGfAfcacgagcuaa usUfsaGfcUfcgugucuAfcUfcgaacsusg SN-255962 ascsacgaGfcUfAfAfgagugaaugu asCfsaUfuCfacucuuaGfcUfcgugususu SN-255973 ascsgagcUfaAfGfAfgugaauguga usCfsaCfaUfucacucuUfaGfcucgusgsu SN-255974 gsasgcuaAfgAfGfUfgaaugugaag csUfsuCfaCfauucacuCfuUfagcucsusu SN-255975 asgscuaaGfaGfUfGfaaugugaaga usCfsuUfcAfcauucacUfcUfuagcuscsg SN-255976 gscsuaagAfgUfGfAfaugugaagau asUfscUfuCfacauucaCfuCfuuagcsusc SN-255977 asgsagugAfaUfGfUfgaagauaaca usGfsuUfaUfcuucacaUfuCfacucususa SN-255978 asgsugaaUfgUfGfAfagauaacaga usCfsuGfuUfaucuucaCfaUfucacuscsu SN-255979 gsasauguGfaAfGfAfuaacagaauu asAfsuUfcUfguuaucuUfcAfcauucsasc SN-255970 asgsgauuAfcAfAfGfcagcaauuua usAfsaAfuUfgcugcuuGfuAfauccususu SN-255971 gscsaauuUfaCfCfUfggaagugaua usAfsuCfaCfuuccaggUfaAfauugcsusu SN-255972 gsasuuugUfgAfUfGfgauuguauau asUfsaUfaCfaauccauCfaCfaaaucsusu SN-255983 gsusgaugGfaUfUfGfuauauuuauu asAfsuAfaAfuauacaaUfcCfaucacsasa SN-255984 gsasuggaUfuGfUfAfuauuuauuua usAfsaAfuAfaauauacAfaUfccaucsasc SN-255985 gscsucaaAfcCfUfCfccuaagacaa usUfsgUfcUfuagggagGfuUfugagcsusu SN-255986 gsascaagCfuGfCfUfgcugugacua usAfsgUfcAfcagcagcAfgCfuugucsusa SN-255987 gsasgcuaGfaUfCfGfuauauuuauu asAfsuAfaAfuauacgaUfcUfagcucsusu SN-255988 gscsauccAfuAfCfUfacagagaaaa usUfsuUfcUfcuguaguAfuGfgaugcsasg SN-255989 gscsaaauGfgAfUfAfguuacaguaa usUfsaCfuGfuaacuauCfcAfuuugcsusu SN-255980 csasaaugGfaUfAfGfuuacaguaaa usUfsuAfcUfguaacuaUfcCfauuugsusu SN-255981 csusguuuCfgUfGfCfaaauauuuau asUfsaAfaUfauuugcaCfgAfaacagsusu SN-255982 gsusuucgUfgCfAfAfauauuuaucu asGfsaUfaAfauauuugCfaCfgaaacsasg SN-255993 gsusgcaaAfuAfUfUfuaucucauua usAfsaUfgAfgauaaauAfuUfugcacsgsa SN-255994 gscsaaauAfuUfUfAfucucauuauu asAfsuAfaUfgagauaaAfuAfuuugcsasc SN-255995 gsasucuaGfuUfCfAfauaaccuaga usCfsuAfgGfuuauugaAfcUfagaucsusu SN-255996 gsusucaaUfaAfCfCfuagaauuuga usCfsaAfaUfucuagguUfaUfugaacsusu SN-255997 csasauaaCfcUfAfGfaauuugaauu asAfsuUfcAfaauucuaGfgUfuauugsusu SN-255998 ascscacaUfaGfCfUfuucaaucugu asCfsaGfaUfugaaagcUfaUfguggususu SN-255999 csasaucuGfuGfCfCfaacaacuaua usAfsuAfgUfuguuggcAfcAfgauugsusu SN-255990 gscscaacAfaCfUfAfuacaauucau asUfsgAfaUfuguauagUfuGfuuggcsusu SN-255991 csasacaaCfuAfUfAfcaauucauca usGfsaUfgAfauuguauAfgUfuguugsusu

[0081] Table 3 Serial 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

[0082] In the modified sequences of this invention, the nucleotide represented by the lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide to its left and right is a 2'-fluoro nucleotide; s indicates that the two adjacent nucleotides are linked by a phosphate dithioester nucleotide bond.

[0083] The nucleic acids described in this invention can be obtained using conventional methods in the art, such as solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis is available through commercial custom services and can therefore be obtained commercially. The modified nucleotide groups can be introduced using nucleotide monomers with corresponding modifications.

[0084] Based on the synthesized nucleic acid (siRNA) as described above, the present invention can further construct shRNA expression plasmids with the same or similar functions as the above nucleic acid. The method for constructing the expression plasmid is well known to those skilled in the art and will not be described in detail here.

[0085] Targeted drug delivery system

[0086] The present invention also provides a targeted drug delivery system comprising a target group, a linker group, and a nucleic acid as described above, which is linked to the target group via the linker group.

[0087] Based on common knowledge in the art, the nucleic acid (siRNA) of this invention exhibits superior inhibitory effects when applied to different targeted drug delivery systems. In other words, the efficacy advantages of the naked sequence and the modified sequence in this invention do not depend on the choice of targeting 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.

[0088] In some specific embodiments, the targeting group can further enhance the targeting of small nucleic acids and can be provided by monosaccharides (e.g., glucose, mannose, allose, alphaose, galactose, galactosamine, N-acetylglucosamine, taloose, fructose, idooose, etc.) and / or peptides (e.g., proteins, monoclonal antibodies, nanobody).

[0089] 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-, and -O-[CH2]m-CONH-[CH2H2O]nO-. Wherein, m and n can each be independently integers from 1 to 10.

[0090] In some embodiments, the targeted drug delivery system includes a ligand and the nucleic acid connected to the ligand, the ligand being 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.

[0091] In some preferred embodiments, the ligand is a GalNAc derivative.

[0092] In some preferred embodiments, the ligand is one or more GalNAc derivatives linked by single-chain, double-chain, or triple-chain branch heads.

[0093] In some further preferred embodiments, the RNAi reagent comprises a compound with the structure shown in Formula I: Formula I

[0094] In the formula, Nu represents the double strand. This targeted drug delivery system utilizes the structural characteristics of 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.

[0095] In practice, under the conditions of coupling reaction and in the presence of coupling reagents, the ligand part can be brought into contact with the nucleotide monomer or the nucleic acid attached to the solid support, thereby connecting the compound part to the nucleic acid via a coupling reaction.

[0096] cell

[0097] The present invention also provides an isolated cell containing the aforementioned nucleic acid.

[0098] In some embodiments, the cells can be used for purposes such as gene function research, disease model research, or drug screening.

[0099] 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.).

[0100] Pharmaceutical Composition

[0101] The present invention also provides a pharmaceutical composition comprising a nucleic acid or targeted drug delivery system as described above and a pharmaceutically acceptable carrier.

[0102] The pharmaceutical composition can be prepared from the nucleic acid and the pharmaceutically acceptable carrier using conventional methods. For example, the pharmaceutical composition can be an injection solution. The injection solution can be used for subcutaneous, intramuscular, or intravenous injection.

[0103] According to the pharmaceutical composition of the present invention, there are no particular requirements for the amount of nucleic acid or targeted drug delivery system and 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 based on nucleic acid), the content of the pharmaceutically acceptable carrier can be 1-100,000 parts by weight (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).

[0104] 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 pH buffer, protectant, and 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 protectant can be at least one of inositol, sorbitol, and sucrose. Based on the total weight of the pharmaceutical composition, the content of the protectant 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.

[0105] 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.

[0106] The dosage of the pharmaceutical composition described in this invention can be a conventional dosage in the art, and the dosage 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 nucleic acid in the pharmaceutical composition.

[0107] Methods and Applications

[0108] The present invention also provides a method for inhibiting the expression of coagulation factor XI in cells, the method comprising: contacting the cells with the nucleic acid, the targeted drug delivery system, or the drug composition to inhibit the expression of coagulation factor XI in the cells.

[0109] In some implementations, the cells are in a subject, for example, a human subject, such as a subject with coagulation factor XI-related disease, or a subject who needs to prevent the risk of coagulation factor XI-related disease.

[0110] In some embodiments, the cells are located in vitro. The method is based on research purposes or for constructing animal models.

[0111] 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%, or 95% (e.g., compared to the expression level of coagulation factor XI before the cells are first contacted with the RNAi reagent or the pharmaceutical composition; e.g., before administering a first dose of the RNAi reagent or the pharmaceutical composition to the subject). In some embodiments, inhibiting the expression of coagulation factor XI reduces the level of coagulation factor XI protein in the subject's serum sample by at least 50%, 60%, 70%, 80%, 90%, or 95%, e.g., compared to the expression level of coagulation factor XI before the cells are first contacted with the RNAi reagent or the pharmaceutical composition.

[0112] The present invention also provides the use of the said nucleic acid, the said targeted drug delivery system, or the said pharmaceutical composition in the treatment and / or prevention of coagulation-related diseases. Specifically, a method for treating and / or preventing coagulation-related diseases includes: administering the said nucleic acid, the said targeted drug delivery system, or the said pharmaceutical composition to a subject.

[0113] The present invention also provides the use of the described nucleic acid, the described targeted drug delivery system, or the described pharmaceutical composition in the preparation of medicaments for the treatment and / or prevention of coagulation-related diseases.

[0114] In some embodiments, the disease is: (i) a disease associated with increased or elevated coagulation factor XI; or (ii) a disease that would benefit from reduced coagulation factor XI expression.

[0115] In some implementations, the disease is selected from diseases related to coagulation disorders (such as an increased risk of thrombosis).

[0116] In some embodiments, the disease is selected from at least one of pulmonary embolism, venous embolism, deep vein embolism, myocardial infarction, and stroke.

[0117] 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.

[0118] In some implementations, the drug can be administered via multiple routes, depending on whether local or systemic treatment is required. Dosage can be referenced as described above and will not be repeated here.

[0119] In some embodiments, administration can be local (e.g., percutaneous patch), pulmonary, such as by inhalation or blowing of powder or spray, including via a 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 intraventricular administration.

[0120] In some embodiments, the nucleic acid, the targeted drug delivery system, or the pharmaceutical composition is administered to the subject via subcutaneous, intravenous, and / or intramuscular administration.

[0121] Example

[0122] 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.

[0123] In the specific embodiments described below, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0124] [Example] [1]

[0125] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 10⁴ Hep3B (Procell, Cat# CL-0102) cells to 96-well cell culture dishes and incubate overnight at 37°C and 5% CO₂. Add RNAiMAX (1.5 μL / well) and small interfering nucleic acids (siRNA) with sequences as shown in Table 2 to Opti-MEM culture medium to make the final concentration per well 1 nM or 10 nM, and continue to culture 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 prepared lysis buffer (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)), mix well, incubate for 10 min, and then add 2.5 μL of stop solution 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 human FXI was Hs01038035_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 (1 loop), 95℃ for 1 second, and 60℃ for 20 seconds (40 loops in total). The real-time fluorescence PCR system used was the QuantStudio™ 6 Pro Real-Time Fluorescence PCR System (Thermo Fisher). FXI gene expression was calculated as 2^-ΔΔCt, with human HPRT1 gene expression used as an internal control. FXI gene expression levels were expressed as a relative value to the control group containing only RNAiMAX. The results are shown in Table 4.

[0126] Table 4. Silent effect of F11 mRNA expression in Hep3B cells compound 1 nM 10 nM compound 1 nM 10 nM compound 1 nM 10 nM 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

[0127] It is evident that siRNAs containing the double-stranded sequence of this invention exhibit varying degrees of inhibitory effects on FXI expression.

[0128] [Example] [2]

[0129] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 10⁴ Hep3B (Procell, Cat# CL-0102) cells to 96-well cell culture dishes and culture overnight at 37°C in a cell culture vessel with 5% CO₂. Add RNAiMAX (1.5 μL / well) and small interfering nucleic acids (siRNA) from Table 5 to Opti-MEM culture medium to make the final concentrations per well 0.0137 nM, 0.0411 nM, 0.1235 nM, 0.3704 nM, 1.111 nM, 3.333 nM, and 10 nM, and continue to culture at 37°C in a cell culture vessel with 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 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 human FXI was Hs01038035_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 loop, followed by 40 loops at 95℃ for 1 second and 60℃ for 20 seconds. The real-time fluorescence PCR system used was a QuantStudio™ 6 Pro Thermo Fisher system. FXI gene expression was calculated as 2^-ΔΔCt, with human HPRT1 gene expression used as an internal control. FXI gene expression levels were expressed relative to the RNAiMAX-only cell group as a control, and the IC50 value was calculated. Results are shown in Table 5.

[0130] Table 5 IC50 values ​​of different siRNAs knocking down F11 expression in Hep3B cells serial number IC50,nM serial 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

[0131] [Example] [3]

[0132] To further verify the activity of siRNA, a siRNA with good activity in Hep3B cells was selected and conjugated with TriGalNAc (the structure of this compound is shown in Formula I). ​​The sequence and conjugation of this targeted drug delivery system are shown in Table 6. On day 0, different human FXI transgenic mice were subcutaneously injected with the targeted drug delivery system shown in Table 6 or PBS (control group). On day 7, the human FXI protein in the blood was tracked and observed, and the results are shown in Figure 1.

[0133] Table 6 serial number Chain of Justice (5'-3') Antisense chain (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

[0134] [Example] [4]

[0135] Add 0.5 mL of cell culture medium (DMEM, 10% fetal bovine serum, 1% penicillin + streptomycin solution) containing 104 Hep3B (Procell, Cat# CL-0102) cells to 96-well cell culture dishes and incubate overnight at 37°C in a 5% CO2 cell culture vessel. Add RNAiMAX (1.5 μL / well) and small interfering nucleic acids (siRNA) as shown in Table 7 to Opti-MEM culture medium to make the final concentration per well 0.4 nM or 2 nM, and continue to culture at 37°C in a 5% CO2 cell culture period for 48 hours. To extract RNA, aspirate the cell culture supernatant, wash with PBS, aspirate again, add 50 μL of prepared lysis buffer (as recommended by the Cells-to-CT kit (ThermoFisher Scientific, Cat#4391851c)), mix well, incubate for 10 min, and then add 2.5 μL of stop solution 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 human FXI was Hs01038035_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 loop, followed by 40 loops at 95℃ for 1 second and 60℃ for 20 seconds. The real-time fluorescence PCR system used was the QuantStudio™ 6 Pro Real-Time Fluorescence PCR System (Thermo Fisher). FXI gene expression was calculated as 2^-ΔΔCt, with human HPRT1 gene expression used as an internal control. FXI gene expression levels were expressed as a relative value to the control group containing only RNAiMAX. The results are shown in Table 8.

[0136] Table 7 serial number Chain of Justice (5'-3') Antisense chain (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

[0137] Table 8 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

[0138] [Example] [5]

[0139] To determine the effects of modifications at different sites on siRNA activity, the siRNAs listed in Table 9 were conjugated with the hepatocyte-targeting compound Tri-GalNAc (its structure is shown in Formula I) and subjected to free uptake experiments in primary monkey hepatocytes. The siRNA-GalNAc samples were dissolved in 100 μL of enzyme-free sterile water to prepare a 10000 μM solution. Then, 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 3-fold diluted at 8 concentration points using PMonH plating medium to obtain the final working solution concentrations of 0.5 μM, 1.4 μM, 4 μM, 12 μM, 37 μM, 111 μM, 333 μM, and 1000 μM. 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 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 cells per well. Prepared sample working solutions were added to the cell solution to achieve final concentrations of 0.5 nM, 1.4 nM, 4 nM, 12 nM, 37 nM, 111 nM, 333 nM, 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, and the plates were washed with 1×PBS buffer. 50 μL of prepared Cells to CT lysis buffer (as recommended by the manufacturer) was added and mixed thoroughly. 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 F11 was Mf02826986_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 (1 loop), 95℃ for 1 second, and 60℃ for 20 seconds (40 loops in total). The real-time fluorescence PCR system used was the QuantStudio™ 6 Pro Real-Time Fluorescence PCR System (Thermo Fisher). F11 gene expression was calculated as 2^-ΔΔCt, and PPIB gene expression was used as an internal control.The F11 gene silencing level was calculated as a percentage compared to the control group containing only culture medium. The siRNA concentration (IC50) that reduced F11 expression by 50% is shown in Table 10.

[0140] Table 9 serial number Chain of Justice (5'-3') Antisense chain (5'-3') SN-685888 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-686683 gsCfsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusu SN-686684 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgUfcuuugUfuGfcaagcsusu SN-686685 gscsuugcAfaCfAfAfagacauuuau-TriGalNAc asUfsaAfaUfgucuuugUfuGfcaagcsusUf

[0141] Table 10 serial number IC50, nM SN-685888 10.66 SN-686683 3.70 SN-686684 3.13 SN-686685 5.81

[0142] [Example] [6]

[0143] SN-685888, which showed good activity in transgenic mice, was selected for further optimization and modification (Table 11). Human FXI transgenic mice were subcutaneously injected with 3 mg / kg of the compound on day 0, and the human FXI protein in the blood was tracked and observed. The results are shown in Figure 2.

[0144] Table 11 serial number Chain of Justice (5'-3') Antisense chain (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

[0145] [Example] [7]

[0146] SN-685299, which showed better in vivo efficacy, was selected to further verify siRNA activity in cynomolgus monkeys. At doses of 5 mg / kg and 20 mg / kg, serum FXI protein levels decreased in a dose-dependent manner, with decreases of approximately 90% and 95%, respectively (Figure 3).

[0147] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the present 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.

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Claims

1. A nucleic acid comprising a sense strand and an antisense strand, wherein the sequence of the sense strand is shown in SEQ ID No. 7; and the sequence of the antisense strand is shown in SEQ ID No.

127.

2. The nucleic acid as described in claim 1, wherein, At least one nucleotide in the nucleic acid is a modified nucleotide or includes a modified interphase bond; the modified nucleotide is selected from one or both of 2'-O-methyl nucleotides and 2'-fluoronucleotides; the modified interphase bond is selected from phosphate thioester nucleotide interphase bonds.

3. The nucleic acid as described in claim 2, wherein, The modified inter-link is selected from one or more of the following: phosphate thioester monoester nucleotide inter-link and phosphate thioester diester nucleotide inter-link.

4. The nucleic acid as described in claim 2, wherein, The antisense strand has 2'-fluoronucleotides at positions 2, 6, 8, 14, 16 and 23 of the nucleotide sequence shown in SEQ ID No. 127, and 2'-O-methylnucleotides at the other positions.

5. The nucleic acid as described in claim 2 or 4, wherein, The positive strand has 2'-fluoronucleotides at positions 2, 7, 9, 10 and 11 of the nucleotide sequence shown in SEQ ID No. 7, and 2'-O-methylnucleotides at the other positions.

6. The nucleic acid as described in claim 2 or 4, wherein, The last 2-4 nucleotides at the 5' end and / or 3' end of the antisense strand contain thiophosphate nucleotide bonds, and the last 2-4 nucleotides at the 5' end and / or 3' end of the sense strand contain thiophosphate nucleotide bonds.

7. The nucleic acid as described in claim 5, wherein, The last 2-4 nucleotides at the 5' end and / or 3' end of the antisense strand contain thiophosphate nucleotide bonds, and the last 2-4 nucleotides at the 5' end and / or 3' end of the sense strand contain thiophosphate nucleotide bonds.

8. The nucleic acid as described in claim 1, wherein, The nucleic acid is selected from any one of the double strands described in SN-255888, SN-256683, SN-256684, SN-256685, SN-255295, SN-255296, SN-255297, SN-255298, and SN-255299; wherein, the sense strand sequence of SN-255888 is 5'-gscsuugcAfaCfAfAfagacauuuau-3', and the antisense strand sequence is 5'-asUfsaAfaUfgucuuugUfuGfcaagcsusu-3'; the sense strand sequence of SN-256683 is 5'-gsCfsuugcAfaCfAfAfagaca The sequence is uuuau-3', and the antisense sequence is 5'-asUfsaAfaUfgucuuugUfuGfcaagcsusu-3'; the sequence is 5'-gscsuugcAfaCfAfAfagacauuuau-3', and the antisense sequence is 5'-asUfsaAfaUfgUfcuuugUfuGfcaagcsusu-3'; the sequence is 5'-gscsuugcAfaCfAfAfagacauuuau-3', and the antisense sequence is 5'-asUfsaAfaUfgucuuugUfuGfcaagcsusUf-3'. The positive chain sequence of SN-255295 is 5'-gsCfsuugcAfaCfAfAfagacauuuau-3', and the negative chain sequence is 5'-asUfsAfaaUfgUfcuuugUfuGfcaagcsusUf-3'; the positive chain sequence of SN-255296 is 5'-gscsuugcAfaCfAfAfaGfacauuuau-3', and the negative chain sequence is 5'-asUfsAfaaUfgUfcuuugUfuGfcaagcsusu-3'; the positive chain sequence of SN-255297 is 5'-gsCfsuugcAfaCfAfAfagacauuuau -3', and the antisense chain sequence is 5'-asUfsAfaaUfgUfcuuugUfuGfcaagcsusu-3'; the positive chain sequence of SN-255298 is 5'-gscsuugcAfaCfAfAfagacauuuau-3', and the antisense chain sequence is 5'-asUfsAfaaUfgUfcuuugUfuGfcaagcsusUf-3'; the positive chain sequence of SN-255299 is 5'-gsCfsuugcAfaCfAfAfagacauuuau-3', and the antisense chain sequence is 5'-asUfsaaaUfgUfcuuugUfuGfcaagcsusUf-3';In each modified sequence, the nucleotide represented by a lowercase letter indicates that the nucleotide is a 2'-O-methyl nucleotide; f indicates that the nucleotide adjacent to it on the left is a 2'-fluoronucleotide; s indicates that the two adjacent nucleotides are linked by a phosphate dithioester nucleotide bond.

9. A targeted drug delivery system comprising a target group, a linker group, and a nucleic acid as described in any one of claims 1 to 8, connected to the target group via the linker group.

10. The targeted drug delivery system as claimed in claim 9, wherein, The targeted drug delivery system includes a ligand and the nucleic acid connected to the ligand, the ligand being 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.

11. The targeted drug delivery system as claimed in claim 10, wherein, The ligand is a GalNAc derivative.

12. The targeted drug delivery system as claimed in claim 11, wherein, The ligands are one or more GalNAc derivatives linked by single-chain, double-chain, or triple-chain branch heads.

13. The targeted drug delivery system as claimed in claim 9, wherein, The structure of the targeted drug delivery system is shown in the following formula: where Nu represents the nucleic acid.

14. An isolated cell containing any one of claims 1 to 8.

15. A pharmaceutical composition comprising any one of claims 1 to 8, or any one of claims 9 to 13, a targeted drug delivery system, and a pharmaceutically acceptable carrier.

16. A method for inhibiting the expression of coagulation factor XI in in vitro cells, the method comprising: The cells are brought into contact with the nucleic acid of any one of claims 1 to 8, the targeted drug delivery system of any one of claims 9 to 13, or the drug composition of claim 15 to inhibit the expression of coagulation factor XI in the cells.

17. Use of a nucleic acid as described in any one of claims 1 to 8, a targeted drug delivery system as described in any one of claims 9 to 13, or a pharmaceutical composition as described in claim 15 in the preparation of a medicament for treating and / or preventing coagulation-related diseases.

18. The use as described in claim 17, wherein, The diseases mentioned are selected from those related to coagulation abnormalities.

19. The use as described in claim 18, wherein, The disease is selected from at least one of pulmonary embolism, venous embolism, deep vein embolism, myocardial infarction, and stroke.

20. The use as described in claim 17, wherein, The nucleic acid, the targeted drug delivery system, or the pharmaceutical composition is administered to the subject via subcutaneous, intravenous, and / or intramuscular administration.

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