Double-stranded ribonucleic acid for reducing expression of blood coagulation factor XI and modifier and application of double-stranded ribonucleic acid

Abstract

The invention provides double-stranded ribonucleic acid for reducing blood coagulation factor XI expression and a modifier and application of the double-stranded ribonucleic acid. Cell experiment results show that the oligonucleotide duplex can significantly inhibit the expression of the FXI gene, and can be used for developing anticoagulant drugs.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine, specifically relating to double-stranded ribonucleic acid (BRNA) that reduces coagulation factor XI expression, its modifications, and their applications. Background Technology

[0002] The coagulation cascade is completed through both intrinsic and extrinsic coagulation pathways. The intrinsic coagulation pathway includes coagulation factors FXI and FXII. In the classical model, upon contact with collagen, RNA, or polyphosphates, FXII is activated. FXIIa further activates downstream FXI, producing FXIa. Through downstream coagulation factors such as FIX and FX, thrombin is ultimately generated via a common pathway. Thrombin initiates fibrin formation while simultaneously activating FXI, creating a positive feedback loop that amplifies the coagulation process. Thrombus formation is primarily triggered by the intrinsic pathway.

[0003] The extrinsic coagulation pathway is typically triggered by tissue factor (TF), which activates FVII and subsequently the common pathway, playing a crucial role in hemostasis. When a blood vessel is damaged, FVII or FVIIa in the blood binds to extravascular tissue factor TF. The FVIIa-TF complex converts FX to FXa, which then converts a limited amount of prothrombin into thrombin. During hemostasis, FXI is converted to FXIa by thrombin, and FXIa subsequently activates FIX and its downstream pathways, leading to clot coagulation, but its overall effect is relatively small. In summary, FXI, a key molecule in the intrinsic coagulation pathway, plays a vital role in regulating the physiological processes of thrombosis, but it is almost entirely absent from pathological processes such as bleeding, which are primarily regulated by extrinsic pathways. Therefore, it could potentially serve as a novel target for antithrombotic therapy.

[0004] Currently marketed anticoagulants include vitamin K antagonists (VKAs), which primarily inhibit prothrombin and FX, and heparin and oral anticoagulants (DOACs), which primarily inhibit FXa and thrombin. These drugs may inhibit hemostasis associated with the extrinsic coagulation pathway while inhibiting thrombus formation, thus posing a certain risk of bleeding during treatment. Conversely, the target FXI in this study has a minimal effect on hemostasis but plays a fundamental role in thrombus formation. Therefore, it can effectively separate the hemostasis and thrombus formation processes, thereby reducing the risk of bleeding while simultaneously providing anticoagulation. Summary of the Invention

[0005] To address the technical problem of the lack of a more effective siRNA duplex for targeting and regulating FXI gene expression in existing technologies, this disclosure provides a siRNA duplex for targeting and regulating FXI gene expression and its use in the prevention and treatment of cardiovascular and cerebrovascular diseases. This disclosure, through modification of the basic siRNA sequence, screens out several siRNA modifiers that significantly inhibit FXI gene expression and provides corresponding siRNA conjugates.

[0006] The technical solutions disclosed herein include, but are not limited to:

[0007] On the one hand, this disclosure provides an siRNA duplex comprising an oligonucleotide duplex composed of a sense strand and an antisense strand paired together.

[0008] On the other hand, this disclosure provides a conjugate for reducing the expression of FXI, comprising the above-mentioned siRNA duplex and a conjugate group attached thereto.

[0009] On the other hand, this disclosure provides a nucleic acid protein complex comprising the double-stranded region of the above-mentioned siRNA double-stranded membrane or the antisense strand of the double-stranded region, and a nuclease.

[0010] On the other hand, this disclosure provides a recombinant vector comprising a nucleic acid molecule encoding the above-mentioned siRNA double strand.

[0011] In some embodiments, the vector backbone of the recombinant vector is selected from recombinant viroid-derived circular RNA vectors, tRNA, rRNA scaffolds, and chimeric tRNA / pre-miRNA vectors.

[0012] On the other hand, this disclosure provides a recombinant cell that synthesizes and secretes the above-mentioned siRNA double strand.

[0013] In some embodiments, the recombinant cells are selected from *Pseudomonas thiophile* and *Corynebacterium glutamicum* deficient in ribonuclease III.

[0014] On the other hand, this disclosure provides a method for preparing siRNA duplexes, including culturing the above-mentioned recombinant cells or chemical synthesis.

[0015] On the other hand, this disclosure provides a pharmaceutical composition comprising the above-described siRNA double strand, the above-described conjugate, or the above-described nucleic acid-protein complex, and a pharmaceutically acceptable carrier.

[0016] On the other hand, this disclosure provides a method for inhibiting FXI gene expression, comprising contacting the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition with target cells.

[0017] In some implementations, the method is for non-diagnostic or non-therapeutic purposes.

[0018] In some implementations, the method is in vivo or in vitro.

[0019] On the other hand, this disclosure provides the use of the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases related to FXI gene expression.

[0020] In some implementations, the FXI gene expression-related diseases are selected from diseases caused by FXI protein overexpression, pathogenic mutations in the FXI gene, abnormal FXI protein metabolism, or abnormal interactions between FXI and another substance.

[0021] In some implementations, the FXI gene expression-related diseases are selected from thrombotic diseases including deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, and thrombosis associated with chronic kidney disease or end-stage renal disease.

[0022] On the other hand, this disclosure provides the use of the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition in the preparation of anticoagulant drugs.

[0023] On the other hand, this disclosure provides the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition or the above-mentioned pharmaceutical composition for the prevention and / or treatment of diseases related to FXI gene expression.

[0024] In some implementations, the FXI gene expression-related diseases are selected from diseases caused by FXI protein overexpression, pathogenic mutations in the FXI gene, abnormal FXI protein metabolism, or abnormal interactions between FXI and another substance.

[0025] In some implementations, the FXI gene expression-related diseases are selected from thrombotic diseases including deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, and thrombosis associated with chronic kidney disease or end-stage renal disease.

[0026] In some implementation plans, the above-mentioned products are used for atrial fibrillation stroke prevention, end-stage renal disease dialysis, and knee replacement anticoagulation.

[0027] On the other hand, this disclosure provides a method for treating diseases related to FXI gene expression, comprising administering an effective amount of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition to a subject in need.

[0028] On the other hand, this disclosure provides the use of the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition in the preparation of drugs for the prevention and / or treatment of cardiovascular and cerebrovascular diseases.

[0029] On the other hand, this disclosure provides the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition for the prevention and / or treatment of cardiovascular and cerebrovascular diseases.

[0030] On the other hand, this disclosure provides a method for preventing and / or treating cardiovascular and cerebrovascular diseases, comprising administering an effective amount of the above-mentioned siRNA double strand, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition to a subject in need. Detailed Implementation

[0031] To make this disclosure easier to understand, certain terms are first defined. Furthermore, it should be noted that whenever a range of values ​​or parameters is enumerated, the purpose is to indicate that intermediate values ​​and ranges of these referenced values ​​are also intended to be part of this disclosure.

[0032] The articles “a” and “an” as used in this article refer to one or more (i.e., at least one) grammatical objects of the article. By way of example, “an element” refers to one element or more elements, such as multiple elements.

[0033] The term “including” is used here to refer to the phrase “including but not limited to” and is used interchangeably with it.

[0034] The term “or” is used here to mean and / or the term “and / or” and is used interchangeably with it, unless the context clearly indicates otherwise.

[0035] As used herein, the term “about” or “approximately” when applied to one or more target values ​​means a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximately” or “about” means a range of values ​​falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0036] As used in this article, "FXI" refers to the gene encoding coagulation factor 11 or the protein expressed by that gene.

[0037] The term "FXI gene" can refer to the wild-type FXI gene or a mutant FXI gene with sequence variations. Many sequence variations in the FXI gene have been identified and can be found in, for example, NCBIdbSNP and UniProt (see, for example, ncbi.nlm.nih.gov / snp).

[0038] “G,” “C,” “A,” and “U” typically represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. “T” and “dT” are used interchangeably herein and refer to deoxyribonucleotides in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it should be understood that the terms “ribonucleotide,” “nucleotide,” or “deoxyribonucleotide” can also refer to a modified nucleotide (as detailed further below) or an alternative substitution. Those skilled in the art will readily recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide with such a substitution). For example, and not limited to, nucleotides containing inosine as a base can pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of this disclosure with a nucleotide containing, for example, inosine. Sequences containing such substitution moieties are applicable to, but are not limited to, double-stranded ribonucleic acids, double-stranded ribonucleic acid modifications, double-stranded ribonucleic acid conjugates, pharmaceutical compositions, and methods of this disclosure.

[0039] The terms “complementary,” “fully complementary,” and “substantially complementary” are used herein to refer to base pairing between the sense and antisense strands of the siRNA, or between the antisense strand of the siRNA and the target sequence, as can be understood from the context in which they are used. In some embodiments herein, a first nucleotide sequence is considered complementary to a second nucleotide sequence if the first nucleotide sequence exhibits sequence complementarity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. In one exemplary embodiment, 18 out of 20 nucleobases of the first nucleotide sequence pair with the corresponding region of the second nucleotide sequence, achieving 90% complementarity.

[0040] The terms “double-stranded ribonucleic acid,” “double-stranded RNA (dsRNA) molecule,” “dsRNA,” and “ribonucleic acid duplex” are used interchangeably to refer specifically to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel, complementary, or substantially complementary nucleic acid strands, with “sense” and “antisense” orientation relative to a target gene, such as the FXI gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi). In this document, in some cases, “siRNA duplex” also typically refers to the technical meaning as defined above.

[0041] As is well known in the art, the terms "siRNA double strand," "double-stranded RNAi agent," "RNAi agent," "small interfering RNA," or "siRNA" refer to small interfering RNA (siRNA) molecules. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silent RNA. siRNA typically comprises a sense strand (also called a guest strand) and an antisense strand (also called a leader strand), each strand being 17 to 30 nucleotides in length, typically 19 to 25 nucleotides in length. The antisense strand is complementary to the target nucleic acid (suitably a mature mRNA sequence) (e.g., at least 95% complementary, e.g., completely complementary), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a double strand or a double-stranded region. The sense and antisense strands of siRNA can form a blunt-ended double strand, or a double strand containing a 3′ overhang, which can be, for example, 1, 2, or 3 nucleotides in length, similar to the products produced by Dicer, which can form RISC substrates in vivo. Efficient extended forms of Dicer substrates have been described in US 8349809 and US 8513207, which are incorporated herein by reference. In some embodiments, both the sense and antisense strands have a 3′ overhang of 2 nucleotides. Thus, the length of the double-stranded region can be, for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides, such as 19, 20, 21, 22, or 23 nucleotides.

[0042] Furthermore, in this document, "siRNA" sometimes also refers to "basic sequence." Specifically, "basic sequence" refers to an siRNA double helix in which each nucleotide is an unmodified nucleotide, and is also referred to as "motif," "siRNA motif," etc., throughout the document. Therefore, in this document, "siRNA," "basic sequence," "motif," and "siRNA motif" are used interchangeably, and their meanings also include the corresponding nucleotide sequence of the siRNA double helix they refer to. Those skilled in the art can clearly understand their precise technical meaning based on the context. Additionally, the 5' terminal nucleotide of the antisense strand of the motif may be linked to a 5' phosphate group or a 5' phosphate-derived group, or may not be linked to a 5' phosphate group or a 5' phosphate-derived group.

[0043] In this document, "siRNA modifier" refers to a double-stranded ribonucleic acid containing at least one modified nucleotide, and in some cases, it is also referred to as "double-stranded ribonucleic acid modifier". In this document, siRNA motifs are modified in different ways to prepare corresponding siRNA modifiers. For example, in some embodiments, the motif is modified using an alternating modification method to obtain alternatingly modified siRNA modifiers. In other embodiments, the motif is modified using a specific modification template to obtain siRNA modifiers modified with that specific modification template. In still other embodiments, the motif is modified using the off-target prevention modification method described herein to obtain off-target prevention modified siRNA modifiers. In some cases, multiple different modification methods can be used simultaneously to modify the same siRNA motif to obtain corresponding siRNA modifiers with multiple modification methods.

[0044] In this document, "siRNA conjugate" refers to a double-stranded ribonucleic acid conjugate or a conjugate of a double-stranded ribonucleic acid modifier obtained by attaching a conjugating group to a double-stranded ribonucleic acid, a double-stranded ribonucleic acid modifier, or a conjugate of a double-stranded ribonucleic acid modifier. Preferably, "siRNA conjugate" refers to a conjugate of a double-stranded ribonucleic acid modifier.

[0045] In some instances herein, "siRNA" refers not only to the unmodified siRNA double strand (or siRNA motif) described above, but may also refer to its corresponding siRNA modifiers and / or siRNA conjugates. For example, in contexts involving, but not limited to, treatment methods and therapeutic agents, siRNA generally refers to at least one of siRNA motifs, siRNA modifiers, and / or siRNA conjugates. The specific technical meaning will be clearly understood by those skilled in the art in the context.

[0046] The term “antisense strand” refers to a strand of double-stranded ribonucleic acid (e.g., RNA double helix in this text) that includes a region substantially complementary to the target sequence. When used herein, the term “complementary region” refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., the target sequence). When the complementary region is not perfectly complementary to the target sequence, mismatches can occur within the molecule or in terminal regions. Typically, the most permissible mismatches are in terminal regions, for example, within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0047] When used herein, the term “sense chain” refers to a double-stranded RNA chain that includes regions substantially complementary to the regions of the antisense chain (as defined herein).

[0048] The term "nonsense sequence" refers to a designed or screened nucleic acid sequence that does not significantly complement the mRNA of any known functional gene in the target organism, and therefore theoretically cannot trigger a specific gene silencing effect. This sequence serves as an experimental control to exclude the influence of non-target factors (such as transfection stress, vector backbone effects, or innate immune responses) on the results of RNA interference experiments.

[0049] The term "alternating modification" refers to a modification method in which nucleotides are modified with 2'-methoxy (2'-OMe) and 2'-fluorine (2'-F) according to the nucleotide sequence of the double-stranded RNA. For example, in the antisense strand of siRNA, odd-numbered positions (i.e., positions 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, and 23) are modified with 2'-methoxy, and even-numbered positions (i.e., positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22) are modified with 2'-fluorine. For the sense strand complementary to this antisense strand, the corresponding position on the antisense strand that is modified with 2'-methoxy is modified with 2'-fluorine; and the complementary pairing position on the sense strand that is modified with 2'-fluorine on the antisense strand is modified with 2'-methoxy. In some implementations, the nucleotides at odd-numbered sites on the sense strand and the nucleotides at even-numbered sites on the antisense strand are modified with 2'-fluorination, while the nucleotides at other sites are modified with 2'-methoxylation.

[0050] For RNA interference (RNAi), the suppression of target genes is achieved by loading the antisense strand of siRNA onto the AGO2 protein and forming a silencing complex (RISC), which then cleaves the gene's transcript mRNA. Loading the RISC requires phosphorylation of the 5' end of the antisense strand. This 5' phosphorylation can occur naturally within the cell via cleavage and polyadenylation factor I subunit 1 (Clp1), or it can be achieved through chemical synthesis. The terms "natural 5' phosphorylation" or "simple and direct 5' phosphorylation" refer to siRNA antisense strand phosphorylation occurring within the cellular environment, rather than through chemical synthesis.

[0051] In this document, "conjugation group" refers to a GalNAc derivative attached to an oligonucleotide. In some cases, the conjugation group includes a targeting group (also referred to as a ligand), and optionally also includes a linker, such as a GalNAc derivative attached to the oligonucleotide via a linker (e.g., a divalent, trivalent, or tetravalent branched linker arm), or, for example, a GalNAc derivative attached to the oligonucleotide via a monovalent linker arm. In most cases, both "ligand" and "conjugation group" have meanings known in the art.

[0052] The term "inhibition" as used herein may be used interchangeably with "reduction," "silence," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. In some instances, "regulation" in this document refers to "inhibition," the specific meaning of which will be readily apparent to those skilled in the art from the context.

[0053] As used herein, the phrase “inhibit FXI expression” includes inhibiting the expression of any FXI gene (such as, for example, the mouse FXI gene, rat FXI gene, monkey FXI gene, or human FXI gene) and variants (e.g., naturally occurring variants) or mutants of the FXI gene. Therefore, the FXI gene can be a wild-type FXI gene, a mutant FXI gene, or, in the case of genetically manipulated cells, cell groups, or organisms, a transgenic FXI gene.

[0054] "Inhibition of FXI gene expression" includes inhibition of the FXI gene at any level, such as at least partial inhibition of FXI gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0055] FXI gene expression can be assessed based on the levels of any variable associated with FXI gene expression, such as FXI mRNA or FXI protein levels. The inhibition can be assessed by a reduction in the absolute or relative level of one or more of these variables compared to a control level. A control level can be any type of control level utilized in the art, such as baseline levels before administration or levels determined from similar untreated or controlled (e.g., buffer-only or inert control) subjects, cells, or samples.

[0056] In this article, in some cases, "regulation" can refer to the same meaning as "inhibition"; correspondingly, "regulating FXI gene expression" can mean "inhibiting FXI gene expression". For those skilled in the art, the specific technical meaning will be clear when combined with the context.

[0057] As used herein, "patient" or "subject" is intended to include humans or non-human animals, preferably mammals such as monkeys. More preferably, the subject or patient is a human.

[0058] As used herein, “FXI-related disease” is intended to include any disease associated with the FXI gene or protein. Such disease can be caused, for example, by overproduction of the FXI protein, mutations in the FXI gene, abnormal cleavage of the FXI protein, or abnormal interactions between FXI and other proteins or other endogenous or exogenous substances. Exemplary FXI-related diseases include thrombotic disorders such as deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, and thrombosis associated with chronic kidney disease or end-stage renal disease.

[0059] As used herein, “therapeutic effective dose” is intended to include the amount of RNAi agent that, when administered to a patient for the treatment of an FXI-related disease, is sufficient to achieve therapeutic effect on the disease (e.g., by attenuating, improving, or maintaining the existing disease or symptoms of one or more diseases). This “therapeutic effective dose” can vary depending on the RNAi agent, how it is administered, the disease and its severity, and the patient’s medical history, age, weight, family history, genetic composition, stage of the pathological process mediated by FXI expression, type of prior or concomitant treatment (if any), and other individual characteristics of the patient to be treated.

[0060] As used herein, a “preventive effective dose” refers to an amount of RNAi agent sufficient to prevent or improve the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced or exhibited symptoms of an FXI-related disease but may be susceptible to it. Improving the disease includes slowing its progression or reducing the severity of subsequent disease development. This “preventive effective dose” can vary depending on the RNAi agent, how it is administered, the risk level for the disease, and the subject’s medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics.

[0061] "Therapeutic effective amount" or "prophylactic effective amount" also includes the amount of RNAi agent that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The RNAi agent used in the methods of this disclosure can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0062] As used herein, the term "sample" includes similar fluids, cells, or tissues isolated from a subject, as well as a collection of fluids, cells, or tissues present in a subject. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. Tissue samples may include samples from tissues, organs, or localized regions. For example, a sample may originate from a specific organ, a portion of an organ, or fluids or cells within those organs. In some embodiments, a sample may originate from the liver (e.g., the entire liver or segments of the liver, or certain types of cells in the liver, such as hepatocytes). In a preferred embodiment, "sample derived from a subject" means blood or plasma drawn from that subject. In other embodiments, "sample derived from a subject" means liver tissue (or a subcomponent thereof) derived from that subject.

[0063] In this article, unless otherwise specified, when referring to any nucleotide position of any strand of siRNA motif, siRNA modifier, siRNA conjugate, siRNA duplex, etc., it means the 5' to 3' orientation.

[0064] In one aspect, this disclosure provides an siRNA double strand comprising a sense strand and an antisense strand forming a reverse complementary double-stranded region, wherein the antisense strand comprises a fragment of at least 15, 16, 17, 18, or 19 consecutive nucleotides or a modified fragment thereof in a sequence as shown in any of SEQ ID NO: 82, 86, 128, 130, 142, 152, 153, and 157.

[0065] The siRNA duplex targets and regulates the expression of the FXI gene, and its target sequence is derived from Homo sapiens coagulation factor XI (FXI) mRNA with accession number NM_000128.4 in the NCBI database.

[0066] In some embodiments, the antisense strand comprises a fragment of at least 17, 18, or 19 consecutive nucleotides or a modified fragment thereof in a sequence such as any of SEQ ID NO: 82, 86, 128, 130, 142, 152, 153, and 157.

[0067] In some embodiments, the positive chain comprises a fragment or modified fragment of at least 15, 16, 17, 18 or 19 consecutive nucleotides in a sequence such as SEQ ID NO: 2, 6, 48, 50, 62, 72, 73 and 77.

[0068] In some implementations, the length of the reverse complementary double-stranded region is 17 to 21 bp, for example, 17, 18 or 19.

[0069] In some embodiments, the lengths of the sense strand and the antisense strand each independently comprise 19-23 nucleotides; preferably, the sense strand comprises 19-21 nucleotides and the antisense strand comprises 21-23 nucleotides.

[0070] In some embodiments, the siRNA duplex comprises any one or a combination of at least two of the following oligonucleotide duplexes composed of positive and negative strands, optionally wherein each of the positive and negative strands independently comprises at least one modified nucleotide:

[0071] (1) The positive chain has a sequence or a fragment thereof as shown in SEQ ID NO: 2; and the negative chain has a sequence or a fragment thereof as shown in SEQ ID NO: 82;

[0072] (2) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 6; and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 86;

[0073] (3) The positive chain has a sequence or a fragment thereof as shown in SEQ ID NO: 48; and the negative chain has a sequence or a fragment thereof as shown in SEQ ID NO: 128;

[0074] (4) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 50; and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 130;

[0075] (5) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 62; and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 142;

[0076] (6) The sense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 72; and the antisense chain has a sequence or a fragment thereof as shown in SEQ ID NO: 152;

[0077] (7) The positive chain has a sequence or a fragment thereof as shown in SEQ ID NO: 73; and the negative chain has a sequence or a fragment thereof as shown in SEQ ID NO: 153;

[0078] (8) The positive chain has a sequence or a fragment thereof as shown in SEQ ID NO: 77; and the negative chain has a sequence or a fragment thereof as shown in SEQ ID NO: 157.

[0079] In some implementations, the siRNA duplex is an RNAi agent used to inhibit the expression of the FXI gene.

[0080] In some implementations, the positive strand differs from any of the sequences in SEQ ID NO: 2, 6, 48, 50, 62, 72, 73 and 7977 by 1-3 nucleotides.

[0081] In some embodiments, the antisense strand differs from any of the sequences in SEQ ID NO: 82, 86, 128, 130, 142, 152, 153 and 157 by 1-3 nucleotides.

[0082] In some implementations, the number of nucleotides in the sense strand may be the same as or different from the number of nucleotides in the antisense strand.

[0083] In some implementations, the sense strand has 19 nucleotides and the antisense strand has 19 nucleotides.

[0084] In some implementations, the sense strand has 19 nucleotides and the antisense strand has 21 nucleotides.

[0085] In some implementations, the sense strand has 20 nucleotides and the antisense strand has 20 nucleotides.

[0086] In some implementations, the sense strand has 20 nucleotides and the antisense strand has 22 nucleotides.

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

[0088] In some implementations, the sense strand has 21 nucleotides and the antisense strand has 23 nucleotides.

[0089] In some implementations, the sense strand has 23 nucleotides and the antisense strand has 23 nucleotides.

[0090] In some implementations, the sense strand and the antisense strand each independently contain at least one modified nucleotide.

[0091] In some embodiments, at least one modified nucleotide is selected from any or a combination of at least two of the following: deoxy-nucleotides, 3'-terminal deoxy-thymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimics.

[0092] In some implementations, at least one strand contains a 3' overhang of at least one nucleotide.

[0093] In some implementations, at least one strand contains a 3' overhang of at least two nucleotides.

[0094] In some implementations, all nucleotides on the sense and antisense strands are modified by modifying the 2' position of the nucleotide ribose.

[0095] In some embodiments, the modification at the 2' position of the nucleotide ribose is selected from any one or a combination of several of the following: 2'-methoxy modification, 2'-methoxyethyl modification, 2'-fluoro modification, 2'-benzyloxy modification, 2'-methylcarbonylamino modification, and 2'-pyridinemethoxy modification.

[0096] In some embodiments, the modification at the 2' position of each nucleotide ribose is selected from a combination of 2'-methoxy modification and 2'-fluoro modification.

[0097] In some embodiments, the modification at the 2' position of each nucleotide ribose is selected from an alternating combination of 2'-methoxy and 2'-fluoro modifications.

[0098] In some implementations, the 2' position of each nucleotide ribose is modified as follows: the odd-numbered positions of the sense strand are all 2'-fluorinated, and the even-numbered positions are all 2'-methoxyinated; and the odd-numbered positions of the antisense strand are all 2'-methoxyinated, and the even-numbered positions are all 2'-fluorinated.

[0099] In some implementations, nucleotides are linked to each other by 3',5'-phosphodiester bonds.

[0100] In some implementations, nucleotides are linked to each other by 3',5'-phosphothioester bonds.

[0101] In some implementations, the aforementioned oligonucleotides have alternating fluorine and oxygen modifications.

[0102] In some embodiments, the first two nucleotides at the 3' and / or 5' ends of the sense and / or antisense strands of the siRNA modifier are connected by a 3',5'-phosphothioester bond; for example, in some embodiments, a chiral pure 3',5'-phosphothioester bond is formed. In some embodiments, one, two, or three 3',5'-phosphothioester bonds may be present between the first four nucleotides at the 5' end of the sense and / or antisense strands, and one, two, or three 3',5'-phosphothioester bonds may be present between the first four nucleotides at the 3' end of the antisense strand. In some embodiments, there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end of the sense strand and between the second and third nucleotides; there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end of the antisense strand and between the second and third nucleotides; and there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 3' end of the antisense strand and between the second and third nucleotides.

[0103] In some embodiments of the present invention, for example, the modification of the 2' position of each nucleotide ribose is as follows: the odd-numbered positions of the sense strand are all 2'-fluorinated, and the even-numbered positions are all 2'-methoxyinated; and the odd-numbered positions of the antisense strand are all 2'-methoxyinated, and the even-numbered positions are all 2'-fluorinated.

[0104] The sense strand contains 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end, as well as between the second and third nucleotides at the 5' end. Similarly, the antisense strand contains 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end, as well as between the second and third nucleotides at the 3' end. Modified siRNA duplexes include, for example, A3-AL, A7-AL, A50-AL, A52-AL, A64-AL, A74-AL, A75-AL, and A79-AL as shown in Table 5.

[0105] This disclosure provides an siRNA conjugate comprising a double-stranded siRNA as described herein and a conjugating group attached to the double-stranded siRNA.

[0106] The double-stranded ribonucleic acid (BRNA) and its modified form disclosed herein can optionally be linked to one or more conjugation groups. The conjugation group can be attached to the sense strand, antisense strand, or both strands at the 3' end, 5' end, or both ends. For example, the conjugation group can be attached to the sense strand. In a preferred embodiment, the conjugation group is attached to the 3' end of the sense strand. In one embodiment, the conjugation group has any GalNAc structure.

[0107] In some implementations, the conjugation group is attached to the 3' or 5' end of the positive strand of the nucleotide.

[0108] In some embodiments, the conjugating group is one or more GalNAc derivatives attached using divalent or trivalent branched connecting arms.

[0109] Typically, the conjugation group comprises at least one pharmaceutically acceptable target group, or further comprises a linker, and the siRNA, the linker, and the target group are sequentially linked. In some embodiments, there are 1-6 target groups. In some embodiments, there are 2-4 target groups. In some embodiments, there are 3 target groups. The conjugation group can be covalently or non-covalently linked to the siRNA molecule, and the linking site can be at the 3' or 5' end of the siRNA's sense strand, at the 5' end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the linking site is at the 3' end of the siRNA's sense strand.

[0110] In some implementations, the pharmaceutically acceptable targeting group may be a conventional ligand in the field of siRNA delivery, such as the various ligands described in WO2009082607A2, which are incorporated herein by reference in their entirety.

[0111] In some embodiments, each ligand is independently selected from a ligand capable of binding to a cell surface receptor. In some embodiments, at least one ligand is capable of binding to a hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is capable of binding to a human hepatocyte surface receptor. In some embodiments, at least one ligand is capable of binding to the liver surface desialylate glycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of ligand-linked siRNA to the target cells.

[0112] In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. That is, after the siRNA molecule forms an siRNA conjugate with a conjugate group containing a galactose or N-acetylgalactosamine molecule as a targeting group, the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4. In some embodiments, when the siRNA is linked to a conjugate group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0113] The targeting group can be linked to the siRNA molecule via a suitable adapter. Those skilled in the art can select a suitable adapter based on the specific type of the targeting group. For details on these adapters, the types of targeting groups, and the connection methods with siRNA, please refer to the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0114] In some embodiments, the structure of the conjugating group is, for example:

[0115] ,

[0116] Wherein, X is a hydroxyl protecting group or H, the hydroxyl protecting group being selected from acetyl, benzoyl or isobutyryl; Y is an amine protecting group or H, the amine protecting group being selected from formyl, acetyl, propionyl, n-butyryl or isobutyryl; n is an integer from 0 to 20; q, r and s are each independently an integer from 1 to 7.

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

[0118] .

[0119] In some embodiments, the conjugating group is, for example:

[0120]

[0121] Where X is oxygen, -N(Y)-, or sulfur;

[0122] Y is C 1-4 Alkyl or C 6-10 Aryl;

[0123] R1 is oxygen or sulfur;

[0124] R2 represents hydrogen, -NH2, or C. 1-4 Alkyl, C 6-10 Aryl, C 1-4 Alkoxy or halogen;

[0125] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d - where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0126] B is -(CH2) e -, where e is an integer from 0 to 7;

[0127] L is either -CONH- or -NHCO-;

[0128] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0129] X2 is -(CH2) g - g is an integer from 1 to 6;

[0130] X3 is oxygen or sulfur;

[0131] Y1 is either 0 or 1;

[0132] Y2 is 0, 1, or 2;

[0133] When Y3 is 1, X4 is CH2; when Y3 is 2, X4 is CH; when Y3 is 3, X3 is carbon.

[0134] m is an integer between 0 and 4;

[0135] n is an integer between 0 and 4.

[0136] In some embodiments, the conjugating group has, for example, any of the following structures:

[0137] ,

[0138] ,

[0139] ,or

[0140] .

[0141] In some embodiments, the siRNA conjugates of this disclosure may have any of the following structures:

[0142] ,

[0143] ,

[0144] ,

[0145] ,or

[0146] .

[0147] In some embodiments, the conjugating group has, for example, the following structure:

[0148]

[0149] Where X is oxygen, -N(Y)-, or sulfur;

[0150] Y is C 1-4 Alkyl or C 6-10 Aryl;

[0151] R1 is oxygen or sulfur;

[0152] R2 represents hydrogen, -NH2, or C. 1-4 Alkyl, C 6-10 Aryl, C 1-4 Alkoxy or halogen;

[0153] A is -(CH2) a -、-(CH2CH2O) b -、-((CH2) c NHCO) d -or-((CH2) c CONH) d- where a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

[0154] B is -(CH2) e -, where e is an integer from 0 to 7;

[0155] L is either -CONH- or -NHCO-;

[0156] X1 is -(CH2) f -or-(CH2CH2O) f CH2-, f is an integer from 1 to 5;

[0157] X2 is -(CH2) g - g is an integer from 1 to 6;

[0158] X3 is oxygen or sulfur;

[0159] Y1 is either 0 or 1;

[0160] Y2 is 0, 1, or 2;

[0161] When Y3 is 1, X4 is CH2; when Y3 is 2, X4 is CH; when Y3 is 3, X3 is carbon.

[0162] m is an integer between 0 and 4;

[0163] n is an integer between 0 and 4;

[0164] q is an integer between 0 and 4.

[0165] In some embodiments, the conjugating group has, for example, any of the following structures:

[0166] , , , ,or .

[0167] In some embodiments, the siRNA conjugates of this disclosure have, for example, any of the following structures:

[0168] ,

[0169] ,

[0170] ,

[0171] ,or

[0172] .

[0173] The sense chain, antisense chain, or either of these chains may be connected to conjugation groups G4, G5, G6, G7, G101, G102, G103, G105, or G106.

[0174] In some embodiments, the conjugation group is attached to the 3' end of the positive chain.

[0175] This disclosure also provides a nucleic acid protein complex comprising the double-stranded region or the antisense strand of the aforementioned double-stranded RNAi agent or siRNA conjugate, and a nuclease.

[0176] In this disclosure, the term "nucleic acid-protein complex" refers to the RNA-induced silencing complex (RISC) formed by the binding of siRNA to Argonaute protein (AGO). The siRNA is then unwound into a sense strand and an antisense strand. The sense strand is degraded, and the antisense strand (guide strand) RISC binds to a target mRNA homologous to the siRNA via base pairing. The RISC functions as a nuclease; the siRNA guides the RISC to cleave the homologous single-stranded mRNA, causing the mRNA to lose its function, i.e., it cannot be translated to produce protein, thus silencing the gene.

[0177] This disclosure also provides a recombinant vector comprising a nucleic acid molecule encoding siRNA as described herein.

[0178] In some embodiments, the vector backbone of the recombinant vector is selected from recombinant viroid-derived circular RNA vectors, tRNA, rRNA scaffolds, and chimeric tRNA / pre-miRNA vectors.

[0179] This disclosure also provides a recombinant cell comprising the aforementioned siRNA or recombinant vector.

[0180] In some embodiments, the recombinant cells are selected from *Pseudomonas thiophilus* and *Corynebacterium glutamicum* deficient in ribonuclease III.

[0181] As used herein, a “recombinant vector” is preferably a vector comprising a regulatory sequence operatively linked to a nucleotide sequence encoding the positive strand contained in the nucleic acid molecule of the present invention. A “recombinant cell” is a cell in which at least one recombinant vector capable of expressing a nucleic acid molecule or at least one strand of such nucleic acid molecule has been introduced.

[0182] This disclosure also provides a method for preparing the siRNA described herein, the method comprising culturing the aforementioned recombinant cells, or directly obtaining the siRNA by chemical synthesis and mixing.

[0183] This disclosure also provides a pharmaceutical composition comprising the said siRNA duplex or corresponding siRNA conjugate, and a pharmaceutically acceptable carrier.

[0184] In one embodiment, a pharmaceutical composition comprising a double-stranded siRNA as described herein and a pharmaceutically acceptable carrier is provided herein. The iRNA-containing pharmaceutical composition may be used to treat or prevent diseases or conditions associated with the expression or activity of the FXI gene, such as atherosclerosis. Such pharmaceutical compositions are formulated based on delivery models. One example is a composition formulated for systemic administration via parenteral delivery, such as subcutaneous injection (SC). Another example is a composition formulated for direct delivery to the brain parenchyma, such as by infusion into the brain, for example, via a continuous pump infusion.

[0185] Pharmaceutical compositions comprising the RNAi agents of this disclosure may be, for example, solutions with or without a buffer solution or compositions containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micellar formulations, emulsions, and gene therapy carriers.

[0186] In the method disclosed herein, the siRNA can be administered in a solution. A free siRNA can be administered in a non-buffered solution, such as physiological saline or water. Alternatively, the free siRNA can also be administered in a suitable buffered solution. The buffer solution may include acetate, citrate, alcohol-soluble gluten, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and volumetric molar osmotic concentration of the buffer containing the siRNA can be adjusted to suit its administration to the subject.

[0187] In some embodiments, the buffer solution further comprises a reagent for controlling the molar osmotic pressure concentration of the solution, such that the molar osmotic pressure concentration is maintained at a desired value, such as the physiological value in human plasma. Solutes that may be added to the buffer solution to control the molar osmotic pressure concentration include (but are not limited to) proteins, peptides, amino acids, non-metabolitic polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is a salt. In some embodiments, the reagent for controlling the molar osmotic pressure concentration of the solution is sodium chloride or potassium chloride.

[0188] The pharmaceutical compositions disclosed herein can be administered at a dose sufficient to inhibit the expression of the FXI gene. Typically, the suitable dose of the siRNA disclosed herein is in the range of about 0.001 to about 200.0 mg per kilogram of body weight per day, generally in the range of about 1 to 50 mg per kilogram of body weight per day. For example, siRNA (e.g., siRNA conjugates) can be administered at doses of about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3. 3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7 6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5 Administer at doses of 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or approximately 50 mg / kg.

[0189] The pharmaceutical composition can be administered once daily, or multiple times at different time intervals from 1 to 365 days, or the siRNA can be administered two, three, or more sub-dose at appropriate intervals within a year, or even administered via continuous infusion or delivery using a controlled-release formulation. In this case, the amount of siRNA contained in each sub-dose must be correspondingly less to achieve the total daily dose. Dosage units can also be compounded for delivery over several days, for example using conventional sustained-release formulations that provide sustained siRNA release over a timeframe of several days. Sustained-release formulations are well known in the art and are particularly useful for delivering reagents at specific sites, thus allowing their use with the reagents disclosed herein. In this embodiment, the dosage unit comprises a corresponding plurality of daily doses.

[0190] In other embodiments, a single dose of the pharmaceutical composition can be administered continuously, with subsequent doses given at intervals of no more than 3, 4, or 5 days, or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of this disclosure, a single dose of the pharmaceutical composition of this disclosure is given weekly. In other embodiments of this disclosure, a single dose of the pharmaceutical composition of this disclosure is given every two months.

[0191] Those skilled in the art will understand that certain factors can influence the dosage and timing required to effectively treat a subject, including (but not limited to) the severity of the disease or condition, previous treatments, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treating a subject with a therapeutically effective dose of the composition may comprise a single treatment or a series of treatments. As described elsewhere herein, the effective dose and in vivo half-life of the individual siRNAs covered by this disclosure can be estimated using conventional methods or based on in vivo testing using suitable animal models.

[0192] Depending on whether local or systemic treatment is desired and depending on the area to be treated, the pharmaceutical compositions of this disclosure can be administered in a variety of ways. Administration can be local (e.g., via a skin patch); pulmonary; such as by inhalation or blowing in a powder or aerosol, including via a nebulizer; intratracheal; intranasal; epidermal; and percutaneous, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, for example, via an implanted device; or intracranial, such as administration within the brain parenchyma, intrasheath, or ventricle.

[0193] The siRNA used in the compositions and methods of this disclosure can be formulated for delivery in a membrane-bound ensemble of molecules, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids disposed in at least one lipid bilayer (e.g., one or more lipid bilayers), having an outer membrane formed of a lipophilic material and an aqueous portion located within. This lipophilic material separates the aqueous interior from the aqueous exterior (although in some instances it may include) of the composition, which typically does not include the siRNA. Liposomes are useful for the transfer and delivery of active ingredients to sites of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is applied to a tissue, the liposome bilayer fuses with the cell membrane bilayer. As the liposome fuses with the cell, the aqueous contents, including the siRNA, are delivered into the cell, where the siRNA can specifically bind to a target RNA and can mediate RNA interference (RNAi). In some cases, these liposomes are also specifically targeted, for example, to guide the siRNA to a specific cell type.

[0194] Liposomes containing siRNA can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent to form micelles. For example, the lipid component can be an amphiphilic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include bile salts, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The siRNA formulation is then added to micelles containing the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the siRNA to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain the corresponding liposome formulation of the siRNA.

[0195] siRNA, such as the RNA duplex disclosed herein, can be encapsulated in lipid formulations (e.g., LNPs or other nucleic acid-lipid particles).

[0196] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. An LNP contains a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit prolonged cycle life after intravenous (iv) injection and accumulate at distal sites (e.g., at sites physically separate from the administration site).

[0197] In one embodiment, the mass ratio of lipids to siRNA duplex is about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0198] In some preferred embodiments, the lipid nanoparticles include cationic lipids, neutral lipids, structural lipids, and polymer-conjugated lipids.

[0199] In some preferred embodiments, the cationic lipid is a compound of formula (I), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight-chain alkyl; L2 is C 12～25 Branched alkyl groups. For example, YK-009 of formula (II) (see patent CN114044741B, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0200] (I)

[0201] (II)

[0202] In some preferred embodiments, the cationic lipid is a compound of formula (II), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein: G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 6~25 Straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 with formula (II-I), YK-402 with formula (II-II), YK-407 with formula (II-III), etc. (see patent CN115784921B, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0203] (II)

[0204] (II-I)

[0205] (II-II)

[0206] (II-III)

[0207] In some preferred embodiments, the cationic lipid is a compound of formula (III), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein: G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight-chain or branched alkyl; R2 is C 12~25 Branched alkyl groups; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-. For example, YK-201 with formula (III-I) structure, YK-202 with formula (III-II) structure, etc. (see patent CN115677518B, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0208] (III)

[0209] (III-I)

[0210] (III-II)

[0211] In some preferred embodiments, the cationic lipid is a compound of formula (IV), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 12~25 Straight-chain or branched alkyl group; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3, or -CH2CH2OH. For example, YK-305 with formula (IV-I) and YK-310 with formula (IV-II) (see patent CN115745820B, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0212] (IV)

[0213] (IV-I)

[0214] (IV-II)

[0215] In some preferred embodiments, the cationic lipid is a compound of formula (V), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein G 1 and G 2 Each is independently unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 OR 5 N, -C (=O) OR 4 -OC(=O)R 4 or -NR 5 C(=O)R 4 ;R 4 For C1-C 12 hydrocarbon group; and R 5 It is an H or C1-C6 hydrocarbon group; for example, ALC0315 of formula (VI) (see patent CN108368028B, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0216] (V)

[0217] (VI)

[0218] In some preferred embodiments, the cationic lipid is a compound of formula (VI), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer, wherein R4 is selected from -(CH2). n Q and -(CH2) n CHQR; Q is selected from the following groups: -OR, -OH, -O(CH2).n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8 and heterocycles; n is 1, 2 or 3; for example, SM102 of formula (VI-I) (see patent application CN110520409A, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0219] (VI)

[0220] (VI-I)

[0221] In some preferred embodiments, the cationic lipid is a compound of formula (VII), or its N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer (see patent CN102625696B, DLIN-MC3-DMA, the entire contents of which are incorporated herein by reference, including, in particular, the general formula and specific compounds therein).

[0222] (VII)

[0223] In some preferred embodiments, the cationic lipid is selected from any one or a combination of at least two of the group consisting of YK-009, YK-401, YK-305, ALC0315, SM102 and DLIN-MC3-DMA.

[0224] In some preferred embodiments, the molar ratio of the cationic lipid to the neutral lipid is 1:1 to 10:1.

[0225] In some preferred embodiments, the molar ratio of the cationic lipid to the structural lipid is 1:1 to 5:1.

[0226] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25~65):(5~25):(25~70):(0.5~5).

[0227] In some preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is (25~65):(5~25):(25~45):(0.5~5).

[0228] In some more preferred embodiments, the molar ratio of the cationic lipid, the neutral lipid, the structural lipid, and the polymer-conjugated lipid is 50:10:38.5:1.5 or 49:10:39.5:1.5.

[0229] In some preferred embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, sterols and their derivatives.

[0230] In some preferred embodiments, the neutral lipid is selected from any one or a combination of at least two of the following: 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 Diether 1,2-Dilinoleoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenoicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoyl 1-Steayl-2-oleoyl-stearoyl-ethanolamine (POPE), 1-stearoyl-2-oleoyl-stearoyl-ethanolamine (DSPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl-phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0231] In some preferred embodiments, the structural lipid is selected from any or at least a combination of the following: cholesterol, nonsterols, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol, tomatine, ursolic acid, α-tocopherol, and corticosteroids.

[0232] In some preferred embodiments, the polymeric conjugated lipid is selected from any or a combination of at least two of the following: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0233] In some preferred embodiments, the polymeric conjugated lipid is selected from any or a combination of at least two of the following: distearylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecylacetamide) (ALC-0159).

[0234] Examples of pharmaceutical compositions disclosed herein include, but are not limited to, aqueous formulations, emulsion formulations, and liposome-containing formulations. These compositions can be derived from a variety of components, including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids. For example, formulations targeting the liver are preferred when treating liver conditions such as liver cancer.

[0235] The pharmaceutical formulations disclosed herein (which can be conveniently presented in unit dosage forms) can be prepared using conventional techniques well known in the pharmaceutical industry. Such techniques include steps such as combining the active ingredients with the drug carrier or excipient. Generally, these formulations are prepared by uniformly and finely combining the active ingredients with a liquid carrier or a finely dispersed solid carrier, or both, and, if necessary, shaping the product.

[0236] The compositions disclosed herein can be formulated into any of a number of possible dosage forms, including, but not limited to, tablets, capsules, gelatin capsules, liquid syrups, soft capsules, suppositories, and enemas. The compositions disclosed herein can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension, such substances including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0237] Some compositions of this disclosure also incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analogue that is inert (i.e., not biologically active in itself) but is considered a nucleic acid in vivo, for example by reducing the bioavailability of biologically active nucleic acids by degrading them or promoting their removal from circulation. Co-administration of nucleic acids and carrier compounds (generally in excess of the latter) can result in a significant reduction in the amount of nucleic acid recovered from the liver, kidneys, or other external circulation reservoirs, presumably due to competition for a common receptor between the carrier compound and the nucleic acid. For example, when co-administered with polyinosinic acid, dextran sulfate, polycytidine, or 2,2'-disulfonic acid 4-acetamido-4'-isothiocyanate, the recovery of partially thiophosphorylated dsRNA in liver tissue can be reduced (Miyao et al., DsRNA Research and Development, 1995, 5, 115-121; Takakura et al., DsRNA & Nucleic Acid Drug Development, 1996, 6, 177-183).

[0238] Compared to carrier compounds, a "drug carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or other arbitrary pharmaceutically inert medium used to deliver one or more nucleic acids to animals. The excipient can be liquid or solid, and when combined with nucleic acids and other components of a particular pharmaceutical composition, the excipient is selected to provide desired volume, consistency, etc., with reference to the intended manner of administration. Typical drug carriers include, but are not limited to: binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate, or dicalcium phosphate); lubricants (e.g., magnesium stearate, talc, silica, colloidal silica, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0239] Pharmaceutically acceptable organic or inorganic excipients that are suitable for non-parenteral administration, do not react toxically with nucleic acids, and are suitable for formulation of the compositions disclosed herein may also be used. Suitable pharmaceutically acceptable carriers include, but are not limited to: water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0240] Formulations for topical administration of nucleic acids may include sterile or non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or nucleic acid solutions in liquid or solid oil matrices. These solutions may also include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration and not toxic to nucleic acids may be used.

[0241] Suitable pharmaceutically acceptable excipients include, but are not limited to: water, salt solutions, alcohols, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0242] This disclosure also provides methods for treating or preventing diseases and conditions that can be regulated by downregulating the expression of the FXI gene. Examples include thrombotic diseases such as deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, and thrombosis associated with chronic kidney disease or end-stage renal disease.

[0243] The siRNA disclosed herein can be administered to a subject using any administration method known in the art, including (but not limited to) subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination thereof. In a preferred embodiment, these agents are administered subcutaneously.

[0244] In another embodiment, siRNA is administered in combination with an additional therapeutic agent. The siRNA and the additional therapeutic agent may be administered in combination in the same composition, for example, parenterally, or the additional therapeutic agent may be administered as part of a separate composition or by another method described herein.

[0245] Other examples of therapeutic agents include those known to treat thrombotic disorders or cardiovascular diseases, including, for example, anticoagulants, antiplatelet agents, and thrombolytic agents for acute myocardial infarction and pulmonary embolism, as well as other drugs. Anticoagulants include warfarin, heparin, low molecular weight heparin (such as enoxaparin), and direct oral anticoagulants (such as dabigatran, rivaroxaban, apixaban, and edoxaban); antiplatelet agents include aspirin, clopidogrel, ticagrelor, and prasugrel; thrombolytic agents for acute myocardial infarction and pulmonary embolism include alteplase, tenecteplase, and streptokinase; other drugs include fondaparinux sodium and bivalirudin.

[0246] In one embodiment, an RNAi agent is given to the patient, and then another therapeutic agent is given to the patient (or vice versa). In another embodiment, the RNAi agent and another therapeutic agent are given simultaneously.

[0247] The "non-diagnostic or non-therapeutic" applications described in this disclosure include, but are not limited to: using them as positive controls to screen for other siRNA duplexes that inhibit FXI gene expression, or using them as a means of inhibition in the laboratory to study the relationship between abnormal FXI gene expression and disease.

[0248] The nucleotide codes used in this article are shown in Table 1 below:

[0249] Table 1

[0250]

[0251] Example

[0252] In the following examples, the p-values ​​for inter-group comparisons were <0.05, indicating statistical significance.

[0253] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0254] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments are commercially available.

[0255] Those skilled in the art will understand that, in the various embodiments of this disclosure, when the test substance in the experiment is an siRNA conjugate, it includes, but is not limited to, inhibition rate, IC50. 50 IC 40 The experimental data and results can reflect the inhibition rate and IC50 of the corresponding siRNA modifier of the siRNA conjugate. 50 IC 40 This presents no obstacle to understanding for those skilled in the art.

[0256] Example 1: The inhibitory effect of siRNA motifs on the FXI gene

[0257] Seventy-eight siRNA motifs were designed and synthesized based on the human FXI mRNA sequence (NM_000128.4), as shown in Table 2. Among them, APC, the base sequence siFXIg1 of the compound siFXIg1M1SP with the highest activity shown in patent CN113227376B, was used as a positive control in this study. ANC, a nonsense sequence, served as a negative control. After transfection into HepG2 cells via lipid nanoparticles (LNPs), the inhibitory effect of each siRNA on the target gene FXI was detected using qPCR and ELISA. The results are shown in Table 4.

[0258] 1.1 Synthesis of siRNA motifs

[0259] Instruments and reagents: CYTiva 192 P model DNA / RNA automated synthesizer, with cross-linked polystyrene beads as the solid carrier, model Primer support 5G Unylinker 350 (Cytiva manufacturer).

[0260] Preparation method:

[0261] The following nucleotide monomer solutions were prepared with acetonitrile at a monomer concentration of 0.15 M: DMT-A-2'-O-TBDMS phosphoramide monomer (Formula 9), DMT-C-2'-O-TBDMS phosphoramide monomer (Formula 10), DMT-G-2'-O-TBDMS phosphoramide monomer (Formula 11) and DMT-U-2'-O-TBDMS phosphoramide monomer (Formula 12).

[0262] Formula 9 Formula 10

[0263] Formula 11 Formula 12

[0264] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using a solid-phase phosphorous acid method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and hydroxyl protection. Specifically, when two nucleotides are linked using a phosphate ester, the linkage of the subsequent nucleoside monomer involves these four steps. When two nucleotides are linked using a phosphate thioester, the linkage of the subsequent nucleoside monomer involves these four steps.

[0265] The preparation process is as follows:

[0266] A solid support was loaded into the designated position of the synthesizer, and the corresponding product was obtained after several synthesis cycles. The synthesis cycle included (1) deprotection, (2) coupling, (3) oxidation / sulfidation and (4) hydroxyl protection. The cycle process and the reagents used are described below:

[0267] (1) Deprotection

[0268] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0269] (2) Coupling

[0270] The acetonitrile solution of each nucleotide monomer was coupled using 0.25 M 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0271] (3) Oxidation / sulfidation

[0272] Oxidation: Oxidation was performed using a 0.05 M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0273] Vulcanization: Vulcanization was carried out using a pyridine solution of 3% hydroxanthin as a vulcanizing agent, followed by rinsing with acetonitrile.

[0274] (4) Hydroxyl protection

[0275] Hydroxyl protection was performed using a 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protecting agent, followed by rinsing with acetonitrile.

[0276] Repeat the above steps in a cyclical manner according to the set nucleotide arrangement order to obtain a product with a specific sequence arrangement of the sense strand or the antisense strand.

[0277] (5) Use 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0278] (6) Ammonolysis and purification

[0279] The reacted solid support was transferred to a reactor, and concentrated ammonia (25-28%, mass percentage) was added. After ammonolysis was maintained at 60°C for 12 h, the system was cooled to room temperature, and the mixture was filtered. The filter cake was washed with a mixture of purified water and ethanol. The filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain the 2'-O-TBDMS protected product.

[0280] (7) Remove TBDMS

[0281] DMSO and triethylamine hydrofluoric acid were added to the obtained product, and the reaction was carried out at 60°C for 2 hours. Then, ammonium acetate aqueous solution was added to the reaction solution, and the mixture was shaken to mix. Anhydrous ethanol was added, and the mixture was shaken to mix. Crystallization was then carried out at -20°C for 8-12 hours. After centrifugation, the supernatant was discarded, and the precipitate was washed with anhydrous ethanol to obtain the unmodified single-chain product.

[0282] (8) Annealing

[0283] The sense and antisense strands of the obtained siRNA motif were mixed in a 1:1 molar ratio, heated to 95°C and held for 3 minutes, and then slowly cooled to room temperature to form a double strand of the siRNA motif.

[0284] Table 2 siRNA motifs

[0285]

[0286]

[0287]

[0288] 1.2 Inhibitory effect of siRNA motif on FXI gene

[0289] 1.2.1 Experimental Methods

[0290] HepG2 cell line was cultured in 10% fetal bovine serum DMEM medium (supplemented with 100× penicillin and 100× streptomycin 10μL / mL) and placed in a cell culture incubator containing 5% CO2 at 37℃.

[0291] Before siRNA transfection of cells, Lipofectamine RNAiMAX (Invitrogen, 13778150) and Opti-MEM were mixed at a volume ratio of 2:98 and vortexed to prepare the transfection mixture. The siRNA compound was diluted to an appropriate concentration using Opti-MEM. For single-site transfection, the siRNA concentration was prepared at 960 pM. 55 μL of the Opti-MEM-diluted siRNA solution was added to 55 μL of the transfection mixture at a 1:1 (v / v) ratio.

[0292] For the blank control group, add 55 μL of the prepared transfection mixture to 55 μL of Opti-MEM. Vortex mix and let stand at room temperature for 15 min.

[0293] Add the prepared transfection reagent to each 24-well cell culture plate (100 μL per well) to achieve a final siRNA concentration of 90 pM per well. Add 500 μL of cell suspension (cell density 1.5 × 10⁻⁶ cells / well). 5( / mL). After mixing using the cross-hatching method, place in a 37°C, 5% CO2 cell culture incubator and incubate for 48 h.

[0294] 48 hours after transfection, RNA was first extracted according to the instructions of the RNA extraction kit (RNeasy Mini Kit, QIAGEN, 74106). Then, the RNA was reverse transcribed into cDNA using the FastKing RT Kit (with gDNase) (TIANGEN, KR116-02). Finally, TB Green was used to transcribe the cDNA. ® The Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara, RR420W (L × 5)) was used for qPCR to quantify FXI mRNA expression levels. The amplification primers used in the qPCR experiment are shown in Table 3. The qPCR reaction program was as follows: heat at 95°C for 30 s, then enter cycling mode, heat at 95°C for 5 s, followed by heating at 60°C for 34 s, for a total of 40 cycles.

[0295] Table 3 qPCR amplification primers

[0296]

[0297] The formula for calculating the FXI mRNA expression rate (%) is as follows:

[0298] Expression rate = (FXI mRNA expression level / FXI mRNA expression level in blank control group) × 100%;

[0299] FXI gene expression inhibition rate = 100% - expression rate (%).

[0300] 1.2.2 Experimental Results

[0301] HepG2 cells were transfected with siRNA compounds using LNP. The inhibition rates of each siRNA motif on FXI mRNA expression after 48 hours are shown in the table below.

[0302] Table 4. Inhibition rate of siRNA motif on FXI gene (%)

[0303]

[0304] Example 2: Inhibitory effect of alternating modified siRNA on the FXI gene

[0305] To improve inhibition rate and stability, the siRNA motifs in Table 2 were modified with alternating 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) nucleotides, and 3',5'-thiophosphate bonds were formed between the nucleotides at the 5' and 3' ends of the antisense strand and the 5' end of the sense strand.

[0306] 2.1 Synthesis of alternating siRNA modifiers

[0307] The alternation modification rules in this disclosure are as follows: nucleotides at odd-numbered sites on the positive strand and even-numbered sites on the negative strand are modified with 2'-F, while nucleotides at other sites are modified with 2'-OMe. Furthermore, there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end of the positive strand, and between the second and third nucleotides at the 5' end; similarly, there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end of the negative strand, and between the second and third nucleotides at the 3' end of the negative strand. siRNAs with alternation modifications designed according to these rules are indicated by adding "-AL" after the original base sequence number, as shown in Table 5.

[0308] Instruments and reagents: Qingke 192 P model DNA / RNA automated synthesizer, with cross-linked polystyrene beads as the solid phase carrier, model Primer support 5G Unylinker 350 (Cytiva).

[0309] For example, the preparation method may include:

[0310] Solutions of the following nucleotide monomers were prepared with acetonitrile at a monomer concentration of 0.15 M: DMT-A-OMe phosphorus amide monomer (Formula 1), DMT-C-OMe phosphorus amide monomer (Formula 2), DMT-G-OMe phosphorus amide monomer (Formula 3), DMT-U-OMe phosphorus amide monomer (Formula 4), DMT-AF phosphorus amide monomer (Formula 5), ​​DMT-CF phosphorus amide monomer (Formula 6), DMT-GF phosphorus amide monomer (Formula 7), and DMT-UF phosphorus amide monomer (Formula 8).

[0311]

[0312]

[0313]

[0314] Nucleoside monomers are linked sequentially from 3' to 5' along the nucleotide arrangement using a solid-phase phosphorous amide method. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or sulfidation, and hydroxyl protection. Specifically, when two nucleotides are linked using a phosphate ester, the linkage of the subsequent nucleoside monomer involves these four steps; when two nucleotides are linked using a phosphate thioester, the linkage of the subsequent nucleoside monomer involves these four steps.

[0315] (1) Deprotection

[0316] The DMT protecting group was removed using a 3% dichloroacetic acid toluene solution as a deprotecting agent, followed by washing with acetonitrile.

[0317] (2) Coupling

[0318] The acetonitrile solution of each nucleotide monomer was coupled using 0.25 M 5-ethylthiotetrazole as an activator, followed by rinsing with acetonitrile.

[0319] (3) Oxidation / sulfidation

[0320] Oxidation: Oxidation was performed using a 0.05 M iodine pyridine / water (90 / 10) solution as the oxidant, followed by rinsing with acetonitrile.

[0321] Vulcanization: Vulcanization was carried out using a pyridine solution of 3% hydroxanthin as a vulcanizing agent, followed by rinsing with acetonitrile.

[0322] (4) Hydroxyl protection

[0323] Hydroxyl protection was performed using a 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protecting agent, followed by rinsing with acetonitrile.

[0324] Repeat the above steps in a cyclical manner according to the set nucleotide arrangement order to obtain a sense or antisense product with a specific sequence arrangement.

[0325] (5) Use 3% dichloroacetic acid toluene solution as a deprotection agent to remove the DMT protecting group of the last nucleotide, and then wash with acetonitrile.

[0326] (6) Ammonolysis and purification

[0327] The solid support was transferred to the reactor, and concentrated ammonia (25%-28%, mass percentage) was added. After ammonolysis was maintained at 60°C for 12 h, the system was cooled to room temperature, and the mixture was transferred to a filter press for filtration. The filter cake was washed with a mixture of purified water and ethanol. The filtrates were combined, passed through a chromatography column, concentrated, and lyophilized to obtain single-chain products modified with 2'-OMe and 2'-F.

[0328] (7) Annealing

[0329] The purified sense and antisense strands were mixed in a 1:1 molar ratio, heated to 95°C and held for 3 min, and then slowly cooled to room temperature to form siRNA duplexes.

[0330] The sense and antisense strands of the siRNAs listed in Table 5 were synthesized according to the above method, resulting in a total of 80 siRNAs. APC-AL was a positive control in this study, consisting of alternating fluorine and oxygen modifications to the base sequence of siFXIg1M1SP (siFXIg1, see Table 2). ANC-AL was a negative control in this study, consisting of alternating modifications to the nonsense sequence ANC (its base sequence is shown in Table 2).

[0331] 2.2 Inhibitory effect of alternating siRNA modifiers on the FXI gene

[0332] On HepG2 cells, the inhibitory effect of the fluorine-oxygen alternating modified siRNA synthesized in Part 2.1 of this example on the FXI gene was determined by qPCR and ELISA, and its dose-dependent effect was evaluated by multi-concentration site transfection experiments.

[0333] 2.2.1 qPCR detection

[0334] Eighty alternating fluorine-oxygen modified sequences (base sequences are shown in Table 2) were subjected to qPCR detection to evaluate their inhibitory efficiency on FXI mRNA expression. The experimental method was as described in 1.2.1 of Example 1.

[0335] The results showed that at a compound transfection concentration of 90 pM, the FXI inhibition rate of APC-AL modified with alternating fluorine and oxygen was 51%, while the inhibition rates of A52-AL and A79-AL were 58% and 52%, respectively, which were superior to those of Yangshen.

[0336] Table 5. Inhibition rate (%) of alternating siRNA modifiers on FXI mRNA

[0337]

[0338]

[0339] 2.2.2 ELISA detection

[0340] To investigate the inhibitory effect of siRNAs with high inhibition rates detected by qPCR on the target gene FXI at the protein level, the supernatant of cells transfected with siRNA for 48 hours was analyzed using the Human Factor XI ELISA kit (Thermo Fisher, EH118RB). The specific procedures are as follows:

[0341] 1) Prepare the diluent and washing buffer in the kit to a 1× concentration using deionized water.

[0342] 2) Prepare standard solutions by serially diluting the standard solution with ELISA diluent at a concentration of 2.5 times.

[0343] 3) Add 100 μL of 5-fold diluted sample, standard and ELISA diluent to the corresponding wells of the antibody-pre-covered 96-well plate, with two replicates for each sample. Cover the plate and incubate at room temperature for 2.5 h.

[0344] 4) Discard the reaction solution and wash 4 times with washing buffer, using 300 μL of washing buffer per well each time.

[0345] 5) Dissolve the detection antibody powder in 100 μL of diluent, and then dilute it 80 times with diluent.

[0346] 6) After washing the plate, add 100 μL of detection antibody to each well, cover the plate, and incubate at room temperature for 1 h.

[0347] 7) Discard the reaction solution and wash 4 times with washing buffer, using 300 μL of washing buffer per well each time.

[0348] 8) Dilute the Streptavidin concentrate 250 times with diluent.

[0349] 9) After cleaning, add 100 μL of Streptavidin solution to each well, cover and incubate at room temperature for 45 min.

[0350] 10) Discard the reaction solution and wash 4 times with washing buffer, using 300 μL of washing buffer per well each time.

[0351] 11) Add 100 μL of TMB solution to each well and incubate at room temperature in the dark for 25 min.

[0352] 12) After incubation, add 50 μL of stop solution to each well.

[0353] 13) Use an ELISA reader to read the absorbance at 450 nm.

[0354] The ELISA data were processed and standardized relative to the transfection reagent control group (expression level relative to the transfection reagent control group = protein expression level of FXI in each sample / protein expression level of FXI in the transfection reagent control group sample).

[0355] The results (Table 6) showed that at a compound transfection concentration of 90 pM, the fluorine-oxygen alternating modified APC-AL of *Codonopsis pilosula* inhibited FXI by 54%, while the inhibition rates of A3-AL and AL52-AL were 57% and 60%, respectively, demonstrating superior efficacy compared to *Codonopsis pilosula*.

[0356] Table 6. Inhibition rate (%) of alternating siRNA modifiers on FXI protein

[0357]

[0358] 2.2.3 IC 50 Detection

[0359] Sixteen sequences that showed high inhibition rates in qPCR and ELISA experiments under single-concentration (90 pM) transfection conditions were transfected at multiple concentrations, and the fitted IC50 was determined. 50 To assess its dose-dependent effect.

[0360] The experimental method was the same as described in 1.2.1, except that eight siRNA transfection sites were performed: 10.0000, 2.5000, 0.6250, 0.1563, 0.0391, 0.0098, 0.0024, and 0.0006 nM. After calculating the inhibition rate at each concentration site, the dose-response curve of the compound was fitted using GraphPad Prism software (four parameter logistic equations), and the IC50 of the compound was calculated. 50 .

[0361] The results (Table 7) show that the IC52-AL, A64-AL, A74-AL, A3-AL, A7-AL, A50-AL, and A75-AL sequences have IC50-AL. 50 The value is superior to that of Yangshen APC-AL.

[0362] Table 7 HepG2 cell line IC50 50 Detection

[0363]

[0364] Notes - b : Yin control, unable to fit IC 50 .

[0365] Example 3: Comparison of inhibition rates of the disclosed siRNA motif with similar sequences in the prior art

[0366] The eight siRNA motifs disclosed herein, namely A3, A7, A50, A52, A64, A74, A75, and A79, exhibited superior in vitro pharmacological activity in Example 2. Six of these siRNA motifs, A3, A50, A64, A74, A75, and A79, are similar to sequences already disclosed in the prior art. This example compares the inhibition rates of the FXI gene using these six sequences at the in vitro cellular level.

[0367] 3.1 Synthesis of siRNA motifs

[0368] Using the synthesis method described in 1.1 of Example 1, the siRNA motifs disclosed in the prior art in Table 8 were synthesized.

[0369] Table 8. Similar sequence information in the prior art

[0370]

[0371] 3.2 Comparison of the inhibitory effects of siRNA motifs on the FXI gene

[0372] The experimental method was as described in 1.2.1 of Example 1, with an siRNA transfection concentration of 90 pM.

[0373] The experimental results are shown in Table 9.

[0374] Table 9. Comparison of in vitro efficacy with publicly disclosed similar sequences.

[0375]

[0376] As shown in the table above, the inhibition rates of the six basic sequences disclosed herein (i.e., A3, A50, A64, A74, A75, and A79) were compared with those of similar motifs in the prior art. The results show that, under the same concentration conditions, the inhibition rates of the above six motifs disclosed herein on FXI mRNA expression in HepG2 cells are superior to those of similar sequences in the prior art.

Claims

1. A siRNA double strand, characterized in that, The siRNA duplex comprises a sense strand and an antisense strand forming an inverse complementary double-stranded region, wherein the antisense strand comprises a fragment of at least 15, 16, 17, 18, or 19 consecutive nucleotides or a modified fragment thereof in any of the sequences shown in SEQ ID NO: 82, 86, 128, 130, 142, 152, 153, and 157.

2. The siRNA double strand as described in claim 1, characterized in that, The antisense strand comprises a fragment of at least 17, 18, or 19 consecutive nucleotides or a modified fragment thereof in a sequence as shown in any of SEQ ID NO: 82, 86, 128, 130, 142, 152, 153, and 157; and / or, the sense strand comprises a fragment of at least 15, 16, 17, 18, or 19 consecutive nucleotides or a modified fragment thereof in a sequence as shown in any of SEQ ID NO: 2, 6, 48, 50, 62, 72, 73, and 77.

3. The siRNA double strand as described in claim 1 or 2, characterized in that, The siRNA duplex satisfies one or more of the following conditions: (1) The length of the reverse complementary double-stranded region is 17~21 bp; (2) The lengths of the sense strand and the antisense strand each independently comprise 19-23 nucleotides; (3) At least one strand contains a 3' overhang of at least one or at least two nucleotides; and, (4) siRNA duplex is an RNAi agent used to inhibit the expression of the FXI gene.

4. The siRNA double strand as described in claim 3, characterized in that, The siRNA duplex comprises any one or a combination of at least two of the following oligonucleotide duplexes composed of selected sense and antisense strands, wherein optionally the sense and antisense strands each independently contain at least one modified nucleotide: (1) The positive chain has a sequence or a fragment thereof as shown in SEQ ID NO: 2; and the negative chain has a sequence or a fragment thereof as shown in SEQ ID NO: 82; (2) The justice chain has a sequence or a fragment thereof as shown in SEQ ID NO: 6; The antisense chain has a sequence or fragment thereof as shown in SEQ ID NO: 86; (3) The justice chain has a sequence or a fragment thereof as shown in SEQ ID NO: 48; The antisense chain has a sequence or fragment thereof as shown in SEQ ID NO: 128; (4) The justice chain has a sequence or a fragment thereof as shown in SEQ ID NO: 50; The antisense chain has a sequence or fragment thereof as shown in SEQ ID NO: 130; (5) The justice chain has a sequence or a fragment thereof as shown in SEQ ID NO: 62; The antisense chain has a sequence or fragment thereof as shown in SEQ ID NO: 142; (6) The justice chain has a sequence or a fragment thereof as shown in SEQ ID NO: 72; The antisense chain has a sequence or fragment thereof as shown in SEQ ID NO: 152; (7) The justice chain has a sequence or a fragment thereof as shown in SEQ ID NO: 73; The antisense chain has a sequence or fragment thereof as shown in SEQ ID NO: 153; (8) The positive chain has a sequence or a fragment thereof as shown in SEQ ID NO: 77; and the negative chain has a sequence or a fragment thereof as shown in SEQ ID NO:

157.

5. The siRNA double strand as described in claim 4, characterized in that, The modified nucleotide is selected from deoxynucleotides, 3'-terminal deoxy-thymidine nucleotides, 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, configuration-restricted nucleotides, restricted ethyl nucleotides, base-free nucleotides, 2'-amino-modified nucleotides, 2'-O-allyl-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-hydroxy-modified nucleotides, 2'-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, morpholinyl nucleotides, aminophosphates, nucleotides containing non-natural bases, tetrahydropyran-modified nucleotides, 1,5-dehydrohexyl-modified nucleotides, cyclohexenyl-modified nucleotides, nucleotides containing thiophosphate groups, nucleotides containing methylphosphate groups, nucleotides containing 5'-phosphate groups, and nucleotides containing 5'-phosphate mimics, as well as combinations thereof.

6. The siRNA double strand as described in claim 5, characterized in that, All nucleotide modifications on the sense and antisense strands are 2'-position modifications of the nucleotide ribose, wherein the 2'-position modification of the nucleotide ribose is selected from any one or a combination of several of 2'-methoxy, 2'-methoxyethyl, 2'-fluoro, 2'-benzyloxy, 2'-methylcarbonylamino and 2'-pyridinemethoxy. And / or, nucleotide monomers are linked to each other by 3',5'-phosphodiester bonds or 3',5'-thiophosphodiester bonds.

7. The siRNA double strand as described in claim 6, characterized in that, The 2' position modification of the nucleotide ribose is selected from an alternating combination of 2'-methoxy and 2'-fluoro. And / or, the first to fourth nucleotides at the 5' end of the sense strand and / or the antisense strand may contain one, two or three 3',5'-phosphothioester bonds, and the first to fourth nucleotides at the 3' end of the antisense strand may contain one, two or three 3',5'-phosphothioester bonds.

8. The siRNA double strand as described in claim 7, characterized in that, The 2' position of the nucleotide ribose is modified as follows: the odd-numbered positions of the sense strand are all 2'-fluorinated, and the even-numbered positions are all 2'-methoxyinated; and the odd-numbered positions of the antisense strand are all 2'-methoxyinated, and the even-numbered positions are all 2'-fluorinated. And / or, there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end of the sense strand and between the second and third nucleotides; there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 5' end of the antisense strand and between the second and third nucleotides; and there are 3',5'-thiophosphate bonds between the first and second nucleotides at the 3' end of the antisense strand and between the second and third nucleotides.

9. A conjugate of siRNA, characterized in that, The conjugate comprises a double-stranded siRNA as described in any one of claims 1-8, and a conjugate group attached thereto.

10. A nucleic acid-protein complex, characterized in that, The nucleic acid-protein complex comprises the double-stranded region of the siRNA double-stranded structure as described in any one of claims 1-8, and a nuclease; or, the nucleic acid-protein complex comprises the antisense strand of the double-stranded region of the siRNA double-stranded structure as described in any one of claims 1-8, and a nuclease.

11. The nucleic acid-protein complex according to claim 10, characterized in that, The nuclease is an AGO protein.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a double-stranded siRNA as described in any one of claims 1-8, a conjugate as described in claim 9, or a nucleic acid-protein complex as described in claim 10 or 11, and a pharmaceutically acceptable carrier.

13. The use of the siRNA duplex as described in any one of claims 1-8, the conjugate as described in claim 9, the nucleic acid-protein complex as described in claim 10 or 11, or the pharmaceutical composition as described in claim 12 in the preparation of a medicament for the prevention or treatment of diseases related to FXI gene expression.

14. The use of the siRNA duplex as described in any one of claims 1-8, the conjugate as described in claim 9, the nucleic acid-protein complex as described in claim 10 or 11, or the pharmaceutical composition as described in claim 12 in the preparation of an anticoagulant.

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