Double-stranded ribonucleic acids reducing coagulation factor xi expression, modifications and uses thereof

Abstract

The present disclosure provides double-stranded ribonucleic acids and modifications and uses thereof for reducing expression of coagulation factor XI. Cell experimental results show that the oligonucleotide duplexes of the present disclosure can significantly inhibit the expression of FXI gene, and can be used for developing anticoagulant drugs.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biological medicine, and particularly relates to double-stranded ribonucleic acid for reducing expression of coagulation factor XI, and modification and application thereof. BACKGROUND

[0002] The coagulation cascade is completed through the endogenous coagulation pathway and the exogenous coagulation pathway. The endogenous coagulation pathway includes coagulation factors FXI and FXII. In the classic model, after contacting collagen, RNA or polyphosphates, FXII is activated, FXIIa further activates downstream FXI to produce FXIa, and through downstream coagulation factors such as FIX and FX, finally generates thrombin through the common pathway, which starts the formation of fibrin and activates FXI, forming a positive feedback to amplify the coagulation process. Thrombosis is mainly triggered by the endogenous pathway.

[0003] The exogenous coagulation pathway is usually initiated by tissure factor (TF), which activates FVII, and then activates the common pathway, and plays an important role in the process of hemostasis. When the blood vessel is damaged, FVII or FVIIa in the blood combines with tissure factor TF from the outside of the blood vessel, and the FVIIa-TF complex converts FX into FXa, and then FXa converts a limited amount of prothrombin into thrombin. In the process of hemostasis, FXI is converted into FXIa by thrombin, and then FXIa activates FIX and its downstream pathway to cause blood clotting, but the overall effect is relatively small. In summary, the important molecule FXI of the endogenous coagulation pathway plays an important role in regulating the physiological process of thrombosis, but almost does not participate in the process of bleeding and other cases mainly regulated by the exogenous pathway, and thus can be used as a new target for antithrombotic therapy.

[0004] Among the currently marketed anticoagulant drugs, vitamin K antagonists (VKA) mainly inhibit prothrombin and FX, heparin and oral anticoagulants (DOAC) mainly inhibit FXa and thrombin. These drugs may inhibit the process of thrombus formation while inhibiting the hemostatic function related to the exogenous coagulation pathway, and thus there is a certain risk of bleeding during treatment. In contrast, the target FXI in this study has little effect on the process of hemostasis, but plays a fundamental role in the process of thrombus formation, and thus can effectively separate the processes of hemostasis and thrombus formation, and thus can reduce the risk of bleeding while anticoagulation. SUMMARY

[0005] To solve the technical problem of lack of a more effective siRNA duplex for targeted regulation of FXI gene expression in the prior art, the present disclosure provides an siRNA duplex for targeted regulation of FXI gene expression and its use in the prevention and treatment of cardiovascular and cerebrovascular diseases. The present disclosure screens a plurality of siRNA modifiers having a significant inhibitory effect on FXI gene expression by modifying the basic sequence of siRNA, and provides corresponding siRNA conjugates.

[0006] The technical solutions of the present disclosure include but are not limited to:

[0007] In one aspect, the present disclosure provides an siRNA duplex comprising an oligonucleotide duplex consisting of a sense strand and an antisense strand.

[0008] In another aspect, the present disclosure provides a conjugate for reducing the expression of FXI, comprising the above-mentioned siRNA duplex, and a conjugate group connected thereto.

[0009] In another aspect, the present disclosure provides a nucleic acid-protein complex comprising the double-stranded region of the above-mentioned siRNA duplex or the antisense strand of the double-stranded region, and a nuclease.

[0010] In another aspect, the present disclosure provides a recombinant vector comprising a nucleic acid molecule encoding the above-mentioned siRNA duplex.

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

[0012] In another aspect, the present disclosure provides a recombinant cell that synthesizes and secretes the above-mentioned siRNA duplex.

[0013] In some embodiments, the recombinant cell is selected from a sulfur-oxidizing Rhodopseudomonas and a ribonuclease III-deficient Corynebacterium glutamicum.

[0014] In another aspect, the present disclosure provides a method for preparing an siRNA duplex, comprising culturing the above-mentioned recombinant cell, or chemical synthesis.

[0015] In another aspect, the present disclosure provides a pharmaceutical composition comprising the above-mentioned siRNA duplex, the above-mentioned conjugate, or the above-mentioned nucleic acid-protein complex, and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure provides a method for inhibiting the expression of an FXI gene, comprising contacting the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid-protein complex, or the above-mentioned pharmaceutical composition with a target cell.

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

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

[0019] In another aspect, the present disclosure provides use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease associated with FXI gene expression.

[0020] In some embodiments, the disease associated with FXI gene expression is selected from the group consisting of overexpression of FXI protein, pathogenic mutation of FXI gene, abnormal metabolism of FXI protein, and disease caused by abnormal interaction of FXI with another substance.

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

[0022] In another aspect, the present disclosure provides use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition in the preparation of an anticoagulant.

[0023] In another aspect, the present disclosure provides use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition for preventing and / or treating a disease associated with FXI gene expression.

[0024] In some embodiments, the disease associated with FXI gene expression is selected from the group consisting of overexpression of FXI protein, pathogenic mutation of FXI gene, abnormal metabolism of FXI protein, and disease caused by abnormal interaction of FXI with another substance.

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

[0026] In some embodiments, the above-mentioned product is used for atrial fibrillation stroke prevention, end-stage kidney disease dialysis, and knee joint replacement anticoagulation.

[0027] In another aspect, the present disclosure provides a method for treating a disease associated with FXI gene expression, comprising administering to a subject in need thereof an effective amount of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein complex, or the above-mentioned pharmaceutical composition.

[0028] In another aspect, the present disclosure provides use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating cardiovascular and cerebrovascular diseases.

[0029] In another aspect, the present disclosure provides use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating cardiovascular and cerebrovascular diseases.

[0030] In another aspect, the present disclosure provides use of the above-mentioned siRNA duplex, the above-mentioned conjugate, the above-mentioned nucleic acid protein composition, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating cardiovascular and cerebrovascular diseases. DETAILED DESCRIPTION

[0031] To make the present disclosure more readily available, certain terms are first defined. In addition, it should be noted that whenever a value or a range of values for a parameter are recited, it is intended that the intermediate values to the recited values are also intended to be part of this disclosure.

[0032] The articles "a" and "an" as used herein mean one or more than one (i.e., at least one) of the grammatical article with the understanding that when there is a plurality of elements, "a" and "an" each means one or more of the plural elements. By way of example, "an element" means one element or one or more elements.

[0033] The term "including" as used herein is intended to mean "including, but not limited to" and is used interchangeably with the phrase "including, but not limited to".

[0034] The term "or" as used herein is intended to mean "and / or" unless otherwise indicated by context.

[0035] As used herein, the term "about" or "approximately," as applied to one or more target values, refers to a value that is similar to the stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from context, the term "approximately" or "about" means a range falling within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (unless such number would exceed 100% of a possible value) of the stated reference value in either direction (greater than or less than).

[0036] As used herein, "FXI" refers to the gene encoding coagulation factor XI or the protein expressed by the gene.

[0037] The term "FXI gene" can be a wild-type FXI gene, or a mutant of the FXI gene that has sequence variations. Numerous sequence variations in the FXI gene have been identified and can be found, for example, in the NCBI dbSNP and UniProt (see, e.g., ncbi.nlm.nih.gov / snp).

[0038] "G," "C," "A," and "U" each generally represent a nucleotide comprising, respectively, guanine, cytosine, adenine, and uracil as the base. "T" and "dT" are used interchangeably herein and refer to a deoxyribonucleotide in which the nucleobase is thymine, such as deoxyribothymine, 2'-deoxythymidine, or thymidine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide (as described further below) or an alternative substituent moiety. The skilled artisan will be well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide, including a nucleotide having such a substituent moiety. For example, without limitation, a nucleotide comprising inosine as its base can pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide comprising uracil, guanine, or adenine can be replaced in a nucleotide sequence of the disclosure by a nucleotide comprising, for example, inosine. Sequences comprising such substituent moieties are suitable for use in, including but not limited to, the double-stranded ribonucleic acids, double-stranded ribonucleic acid modifications, double-stranded ribonucleic acid conjugates, pharmaceutical compositions, and methods of the disclosure, among others.

[0039] The terms "complementary," "fully complementary," and "substantially complementary" can be used herein to refer to base pairing between the sense strand and the antisense strand of an siRNA, or between the antisense strand of an siRNA and a target sequence, as will be understood from the context of their use. In some aspects herein, a first nucleotide sequence can be considered complementary to a second nucleotide sequence if the first nucleotide sequence exhibits at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% degree of sequence complementarity with the second nucleotide sequence. In an exemplary embodiment, 18 of 20 nucleobases of the first nucleotide sequence pair with the corresponding region of the second nucleotide sequence, achieving 90% complementarity.

[0040] The terms "double-stranded ribonucleic acid," "double-stranded RNA (dsRNA) molecule," "dsRNA," "ribonucleic acid duplex," can be used interchangeably and refer specifically to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel, complementary or substantially complementary nucleic acid strands having "sense" and "antisense" orientation with respect to a target gene, e.g., FXI gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of a target RNA, e.g., mRNA, through a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi. In this context, "siRNA duplex" also typically refers to the technical meaning indicated by the above definition in some cases.

[0041] As is well known in the art, the term "siRNA duplex," "double-stranded RNAi agent," "RNAi agent," "small interfering ribonucleic acid," or "siRNA" refers to a small interfering ribonucleic acid RNAi molecule. It is a class of double-stranded RNA molecules, also known in the art as short interfering RNA or silencing RNA. The siRNA typically comprises a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand), each strand being 17 to 30 nucleotides in length, typically 19 to 25 nucleotides in length, wherein the antisense strand is complementary (such as at least 95% complementary, such as fully complementary) to a target nucleic acid (suitably a mature mRNA sequence), and the sense strand is complementary to the antisense strand, such that the sense and antisense strands form a duplex or duplex region. The sense and antisense strands of the siRNA can form a blunt-end duplex, or can form a duplex comprising a 3' overhang, which can be, for example, 1, 2, or 3 nucleotides in length, similar to the product of Dicer production, which can form a RISC substrate in vivo. Efficient extensions of the Dicer substrate have been described in US 8349809 and US 8513207, incorporated herein by reference. In some embodiments, both the sense and antisense strands have a 3' overhang of 2 nucleotides in length. Thus, the duplex region can be, for example, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, such as 19, 20, 21, 22, or 23 nucleotides in length.

[0042] Also, in the present context, "siRNA" in some cases also refers to "base sequence". In the present context, "base sequence" in some cases specifically refers to an siRNA duplex in which each nucleotide in the double-stranded ribonucleic acid is an unmodified nucleotide, also appearing throughout the text as "motif", "siRNA motif", etc. Thus, in the present context, "siRNA", "base sequence", "motif", "siRNA motif" can be used interchangeably, and their meaning also includes the corresponding nucleotide arrangement order of the referred siRNA duplex. In the present context, the skilled person can clearly understand the exact technical meaning referred to by them according to the technical meaning of the context. In addition, the 5' terminal nucleotide of the antisense strand of the motif can be linked with a 5' phosphate group or a 5' phosphate derivative group or can not be linked with a 5' phosphate group or a 5' phosphate derivative group.

[0043] In the present context, "siRNA modifier" refers to a double-stranded ribonucleic acid comprising at least one modified nucleotide, in some cases also appearing as "double-stranded ribonucleic acid modifier". In the present context, different modifications of the siRNA motif are made to prepare the corresponding siRNA modifier. For example, in some embodiments, the motif is modified using an alternating modification to obtain an alternating modified siRNA modifier. In other embodiments, the motif is modified using a specific modification template modification to obtain a specific modification template modified siRNA modifier. In yet other embodiments, the motif is modified using an off-target prevention modification in the present context to obtain an off-target prevention modified siRNA modifier. In some cases, multiple different modifications can be used to modify the same siRNA motif to obtain the corresponding siRNA modifier with multiple modifications.

[0044] In the present context, "siRNA conjugate" refers to a conjugate of a double-stranded ribonucleic acid conjugate or a double-stranded ribonucleic acid modifier obtained by linking a conjugate group to the double-stranded ribonucleic acid, the double-stranded ribonucleic acid modifier. Preferably, "siRNA conjugate" refers to a conjugate of a double-stranded ribonucleic acid modifier.

[0045] In some cases in the present context, "siRNA" not only refers to the unmodified siRNA duplex (or siRNA motif) described above, but can also refer to the corresponding siRNA modifier and / or siRNA conjugate, for example, in the context of including but not limited to therapeutic methods, therapeutic agents, etc., siRNA refers to at least one of siRNA motif, siRNA modifier and / or siRNA conjugate. For the skilled person, the specific technical meaning referred to by them can be clearly understood in combination with the context.

[0046] The term "antisense strand" refers to the strand of a double-stranded ribonucleic acid (e.g., an RNA duplex herein) that includes a region of substantial complementarity to a target sequence. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence defined herein (e.g., a target sequence). When the region of complementarity is not perfectly complementary to the target sequence, mismatches can be internal or at the terminal regions of the molecule. Generally, the most tolerated mismatches are at the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' terminus.

[0047] The term "sense strand" as used herein refers to the strand of a double-stranded ribonucleic acid that includes a region of substantial complementarity to a region of an antisense strand (as the term is defined herein).

[0048] The term "non-sense sequence" is a nucleic acid sequence that is artificially designed or screened to not bind significantly complementary to the mRNA of any known functional gene in the target organism, and thus theoretically cannot trigger specific gene silencing effect. The sequence is used as an experimental control to exclude the influence of non-target factors (such as transfection stress, vector backbone effect, or innate immune response) on the results in the RNA interference experiment.

[0049] The term "alternating modification" refers to the modification of nucleotides with 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F) modifications in an alternating pattern along the sequence of the nucleotides of a double-stranded ribonucleic acid. For example, for the antisense strand of an siRNA, the odd-numbered positions (i.e., positions 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23) are modified with 2'-OMe and the even-numbered positions (i.e., positions 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22) are modified with 2'-F. For the sense strand that is complementary to the antisense strand, the positions on the sense strand that correspond to the 2'-OMe modified positions on the antisense strand are modified with 2'-F and the positions on the sense strand that correspond to the 2'-F modified positions on the antisense strand are modified with 2'-OMe. In some embodiments, the odd-numbered positions of the sense strand are modified with 2'-F and the even-numbered positions of the sense strand are modified with 2'-OMe.

[0050] For RNA interference (RNAi), inhibition of a target gene is achieved by loading of the antisense strand of an siRNA by the AGO2 protein and assembly of a silencing complex (RISC) that cleaves the transcript mRNA of the gene. Loading of the silencing complex (RISC) requires 5'-phosphorylation of the antisense strand. 5'-terminal phosphorylation can occur naturally in the cell by cleavage and polyadenylation factor I subunit 1 (Clp1) or can be achieved by chemical synthesis. The term "natural 5'-terminal phosphorylation" or "simple direct 5'-terminal phosphorylation" refers to 5'-terminal phosphorylation of the antisense strand of an siRNA that is completed in the cellular environment and not by chemical synthesis.

[0051] In the present context, a "conjugate group" is a GalNAc derivative attached to an oligonucleotide. In some cases, a conjugate group comprises a targeting group (also referred to as a ligand), optionally further comprising a linker, e.g. a GalNAc derivative linked to an oligonucleotide via a linker (e.g. a divalent, trivalent or tetravalent branched linking arm), and further e.g. a GalNAc derivative attached to an oligonucleotide via a monovalent linking arm. In most cases, "ligand" and "conjugate group" have the meaning well known in the art.

[0052] The term "inhibit", as used herein, can be used interchangeably with "reduce", "silence", "down-regulate", "suppress" and other similar terms, and includes inhibition at any level. In the present context, "inhibit" in some cases refers to the meaning of "reduce", and the skilled person will be aware of the specific meaning referred to depending on the context.

[0053] As used herein, the phrase "inhibiting expression of FXI" includes inhibiting expression of any FXI gene (such as e.g. a mouse FXI gene, a rat FXI gene, a monkey FXI gene, or a human FXI gene), as well as variants (e.g. naturally occurring variants) or mutants of FXI genes. Thus, the FXI gene can be a wild-type FXI gene, a mutant FXI gene, or a transgenic FXI gene in the context of a genetically manipulated cell, cell population, or organism.

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

[0055] Expression of the FXI gene can be assessed based on any variable level associated with FXI gene expression, e.g., FXI mRNA level or FXI protein level. The inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level utilized in the art, e.g., a pre-dose baseline level or a level determined from a similar untreated or control (e.g., buffer control or inert agent control) treated subject, cell, or sample.

[0056] In some instances herein, "modulating" can refer to the same meaning as "inhibiting"; accordingly, "modulating FXI gene expression" can mean "inhibiting FXI gene expression". The specific technical meaning will be clear to one of skill in the art in context.

[0057] As used herein, "patient" or "subject" is intended to include a human or non-human animal, preferably a mammal, e.g., a monkey. 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 a disease can be caused, e.g., by excess production of the FXI protein, by mutation of the FXI gene, by abnormal cleavage of the FXI protein, by abnormal interaction between FXI and other proteins or other endogenous or exogenous substances. Exemplary FXI-related diseases include deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, thrombotic disease including thrombosis associated with chronic kidney disease or end-stage kidney disease.

[0059] As used herein, "therapeutically effective amount" is intended to include the amount of an RNAi agent that, when administered to a patient for treating a FXI -associated disease, is sufficient to effect treatment as defined by partial or complete alleviation of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of the disease). The "therapeutically effective amount" can vary depending on the RNAi agent, how the agent is administered, the disease and its severity, and the history, age, weight, family history, genetic makeup, stage of the pathological process mediated by FXI expression, type of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

[0060] As used herein, "prophylactically effective amount" refers to an amount of an RNAi agent that, when administered to a subject who does not yet experience or exhibit symptoms of a FXI -associated disease, but who is susceptible to or otherwise at risk for the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the progression of the disease or reducing the severity of the disease that develops later. The "prophylactically effective amount" can vary depending on the RNAi agent, how the agent is administered, the degree of risk for the disease, and the history, age, weight, family history, genetic makeup, type of previous or concomitant treatments, if any, and other individual characteristics of the subject susceptible to the disease.

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

[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 plasma, cerebrospinal fluid, ocular fluid, lymphatic fluid, urine, saliva, and the like. Tissue samples can include samples from a tissue, organ or a localized region. For example, a sample can be derived from a particular organ, organ portion, or fluids or cells within these organs. In certain embodiments, a sample can be derived from the liver (e.g., the entire liver or certain segments of the liver, or certain types of cells in the liver, e.g., hepatocytes). In preferred embodiments, a "sample derived from a subject" refers to blood or plasma drawn from the subject. In other embodiments, a "sample derived from a subject" refers to liver tissue (or sub-components thereof) derived from the subject.

[0063] In this document, when referring to any nucleotide position of any strand of an siRNA motif, siRNA modifier, siRNA conjugate, siRNA duplex, etc., the 5' to 3' direction is intended, unless otherwise specified.

[0064] In one aspect, the present disclosure provides an siRNA duplex comprising a sense strand and an antisense strand forming a reverse complementary duplex region, the antisense strand comprising a fragment of at least 15, 16, 17, 18, or 19 contiguous nucleotides in the sequence as set forth in any one of SEQ ID NOs: 82, 86, 128, 130, 142, 152, 153, and 157, or a modified fragment thereof.

[0065] The siRNA duplex targets the regulation of FXI gene expression, the target sequence 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 contiguous nucleotides in the sequence as set forth in any one of SEQ ID NOs: 82, 86, 128, 130, 142, 152, 153, and 157, or a modified fragment thereof.

[0067] In some embodiments, the sense strand comprises a fragment of at least 15, 16, 17, 18, or 19 contiguous nucleotides in the sequence as set forth in any one of SEQ ID NOs: 2, 6, 48, 50, 62, 72, 73, and 77, or a modified fragment thereof.

[0068] In some embodiments, the reverse complementary duplex region has a length of 17-21 bp, for example, 17, 18, or 19.

[0069] In some embodiments, the sense strand and antisense strand independently comprise 19-23 nucleotides in length, respectively; preferably, the sense strand comprises 19-21 nucleotides, and the antisense strand comprises 21-23 nucleotides.

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

[0071] (1) the sense strand has the sequence as set forth in SEQ ID NO: 2, or a fragment thereof; and the antisense strand has the sequence as set forth in SEQ ID NO: 82, or a fragment thereof;

[0072] (2) the sense strand has the sequence as set forth in SEQ ID NO: 6, or a fragment thereof; and the antisense strand has the sequence as set forth in SEQ ID NO: 86, or a fragment thereof;

[0073] (3) the sense strand has the sequence as shown in SEQ ID NO: 48, or a fragment thereof; and the antisense strand has the sequence as shown in SEQ ID NO: 128, or a fragment thereof;

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

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

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

[0077] (7) the sense strand has the sequence as shown in SEQ ID NO: 73, or a fragment thereof; and the antisense strand has the sequence as shown in SEQ ID NO: 153, or a fragment thereof;

[0078] (8) the sense strand has the sequence as shown in SEQ ID NO: 77, or a fragment thereof; and the antisense strand has the sequence as shown in SEQ ID NO: 157, or a fragment thereof.

[0079] In some embodiments, the siRNA duplex is an RNAi agent for inhibiting expression of a FXI gene.

[0080] In some embodiments, the sense strand differs from any one of SEQ ID NOs: 2, 6, 48, 50, 62, 72, 73, and 77 by 1-3 nucleotides.

[0081] In some embodiments, the antisense strand differs from any one of SEQ ID NOs: 82, 86, 128, 130, 142, 152, 153, and 157 by 1-3 nucleotides.

[0082] In some embodiments, the sense strand has the same number of nucleotides as the antisense strand or a different number of nucleotides.

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

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

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

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

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

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

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

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

[0091] In some embodiments, the at least one modified nucleotide is selected from any one or a combination of at least two of the group consisting of: a deoxy-nucleotide, a 3' terminal deoxy-thymine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a restricted ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-0-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

[0092] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide.

[0093] In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0094] In some embodiments, the modification of all nucleotides on the sense strand and the antisense strand is a modification of the 2' position of the ribose of the nucleotide.

[0095] In some embodiments, the modification at the 2' position of the ribose of each nucleotide is selected from the group consisting of a 2'-methoxy modification, a 2'- methoxyethyl modification, a 2'-fluoro modification, a 2'-benzyloxy modification, a 2'- methylcarbonylamino modification, and a 2'-pyridyloxy modification.

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

[0097] In some embodiments, the modification at the 2' position of the ribose of each nucleotide is selected from the group consisting of a 2'-methoxy modification and a 2'- fluoro modification.

[0098] In some embodiments, the modification at the 2' position of the ribose of each nucleotide is selected from the group consisting of a 2'-methoxy modification and a 2'- fluoro modification.

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

[0100] In some embodiments, the nucleotides are linked to each other by 3',5'- phosphodiester bonds.

[0101] In some embodiments, the foregoing oligonucleotide has alternating fluorine and oxygen modifications.

[0102] In some embodiments, the 3' terminal and / or 5' terminal first 1-2 nucleotides of the sense strand and / or the antisense strand of the siRNA modification are linked by 3',5'- phosphorothioate bonds, e.g., in some embodiments, chirally pure 3',5'- phosphorothioate bonds are formed. In some embodiments, the 5' terminal first 1-4 nucleotides of the sense strand and / or the 3' terminal first 1-4 nucleotides of the antisense strand can comprise 1, 2, or 3 3',5'-phosphorothioate bonds. In some embodiments, the 3',5'- phosphorothioate bonds are present between the 5' terminal first and second nucleotides and between the second and third nucleotides of the sense strand; the 3',5'- phosphorothioate bonds are present between the 5' terminal first and second nucleotides and between the second and third nucleotides of the antisense strand, and the 3',5'- phosphorothioate bonds are present between the 3' terminal first and second nucleotides and between the second and third nucleotides of the antisense strand.

[0103] In some embodiments of the application, the 2' position of each ribose nucleotide is modified in the following manner: the odd-numbered positions of the sense strand are 2'-fluoro modified and the even-numbered positions are 2'-methoxy modified; and the odd-numbered positions of the antisense strand are 2'-methoxy modified and the even-numbered positions are 2'-fluoro modified;

[0104] the first and second nucleotides from the 5' end of the sense strand and between the second and third nucleotides from the 5' end of the sense strand are linked by 3',5'- phosphorothioate linkages; and the first and second nucleotides from the 5' end of the antisense strand and between the second and third nucleotides from the 5' end of the antisense strand are linked by 3',5'- phosphorothioate linkages, and the first and second nucleotides from the 3' end of the antisense strand and between the second and third nucleotides from the 3' end of the antisense strand are linked by 3',5'- phosphorothioate linkages. 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] The present disclosure provides an siRNA conjugate comprising an siRNA duplex as described herein and a conjugate group linked to the siRNA duplex.

[0106] The double stranded ribonucleic acid, double stranded ribonucleic acid modification of the present disclosure can be optionally linked to one or more conjugate groups. The conjugate group can be attached to the sense strand, the antisense strand, or both strands at the 3' end, the 5' end, or both ends. For example, the conjugate group can be linked to the sense strand. In preferred embodiments, the conjugate group is linked at the 3' end of the sense strand. In one embodiment, the conjugate group has any GalNAc structure.

[0107] In some embodiments, the conjugate group is linked at the 3' end or the 5' end of the nucleotide sense strand.

[0108] In some embodiments, the conjugate group is one or more GalNAc derivatives attached with a bivalent or trivalent branched linker arm.

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

[0110] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand conventional in the art of siRNA delivery, such as the various ligands described in WO2009082607A2, incorporated herein by reference in its 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 a ligand capable of binding to a liver cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a human liver cell surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to a liver surface asialoglycoprotein receptor (ASGPR). The classes of such ligands are well known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of the target cell, mediating delivery of the siRNA linked to the ligand to the target cell.

[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 a siRNA conjugate with the conjugation group containing the galactose or N-acetylgalactosamine molecule as the targeting group, the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4. In some embodiments, when the siRNA is linked to the conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0113] The targeting group can be linked to the siRNA molecule via a suitable linker, which can be selected by one skilled in the art according to the specific type of the targeting group. The classes of such linkers, targeting groups, and the manner of linking to the siRNA can be found in the disclosure of WO2015006740A2, incorporated herein by reference in its entirety.

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

[0115]

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

[0117] ​In some embodiments, the conjugate group has the structure:

[0118] .

[0119] In some embodiments, the conjugate group has the structure:

[0120]

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

[0122] Y is C 1-4 alkyl or C 6-10 aryl;

[0123] R1is oxygen or sulfur;

[0124] R2is hydrogen, -NH2, 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 -, wherein a is an integer from 1 to 15, b is an integer from 1 to 7, c is an integer from 1 to 7, and d is an integer from 1 to 5;

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

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

[0128] X1is -(CH2) f - or -(CH2CH2O) f CH2-, and f is an integer from 1 to 5;

[0129] X2is -(CH2) g -, and g is an integer from 1 to 6;

[0130] X3is oxygen or sulfur;

[0131] Y1is 0 or 1;

[0132] Y2is 0, 1 or 2;

[0133] X4is CH2when Y3is 1, CH when Y3is 2, and carbon when Y3is 3;

[0134] m is an integer from 0 to 4;

[0135] n is an integer from 0 to 4.

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

[0137] ,

[0138] ,

[0139] , or

[0140] .

[0141] In some embodiments, the siRNA conjugate of the present disclosure can have any of the following structures:

[0142] ,

[0143] ,

[0144] ,

[0145] , or

[0146] .

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

[0148]

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

[0150] Y is C 1-4 alkyl or C 6-10 aryl;

[0151] R1is oxygen or sulfur;

[0152] R2is hydrogen, -NH2, 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) dwherein 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 wherein e is an integer from 0 to 7;

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

[0156] X1is -(CH2) f - or -(CH2CH2O) f CH2-, and f is an integer from 1 to 5;

[0157] X2is -(CH2) g -, and g is an integer from 1 to 6;

[0158] X3is oxygen or sulfur;

[0159] Y1is 0 or 1;

[0160] Y2is 0, 1, or 2;

[0161] X4is CH2when Y3is 1; CH when Y3is 2; and carbon when Y3is 3;

[0162] m is an integer from 0 to 4;

[0163] n is an integer from 0 to 4;

[0164] q is an integer from 0 to 4.

[0165] In some embodiments, the siRNA conjugate of the present disclosure has, for example, any one of the following structures:

[0166] , , , , or .

[0167] In some embodiments, the siRNA conjugate of the present disclosure has, for example, any one of the following structures:

[0168] ,

[0169] ,

[0170] ,

[0171] , or

[0172] .

[0173] wherein either or both of the sense strand, the antisense strand can be linked to conjugate groups G4, G5, G6, G7, G101, G102, G103, G105, or G106.

[0174] In some embodiments, the conjugate group is linked at the 3' end of the sense strand.

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

[0176] In the present disclosure, the term "nucleic acid protein complex" refers to the binding of siRNA to Argonaute protein (AGO) to form an induced silencing complex (RISC). The siRNA is then unwound into a sense strand and an antisense strand. The sense strand is degraded and the antisense strand (guide strand) RISC binds to the target mRNA homologous to the siRNA through base pairing. RISC has the function of a nuclease, and siRNA guides RISC to cut the homologous single-stranded mRNA, resulting in the loss of function of the mRNA, i.e. unable to translate to produce a protein, that is, to "silence" the gene.

[0177] The present disclosure also provides a recombinant vector comprising a nucleic acid molecule encoding the siRNA as disclosed.

[0178] In some embodiments, the vector backbone of the recombinant vector is selected from the group consisting of a recombinant virus-like derived circular RNA vector, a tRNA, a rRNA scaffold, and a chimeric tRNA / pre-miRNA vector.

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

[0180] In some embodiments, the recombinant cell is selected from the group consisting of a Sulfolobus acidocaldarius and a ribonuclease III-deficient Corynebacterium glutamicum.

[0181] As used herein, a "recombinant vector" is preferably a vector comprising regulatory sequences operably linked to a nucleotide sequence encoding the sense strand comprised in the nucleic acid molecule of the present application. A "recombinant cell" is a cell in which at least one recombinant vector has been introduced which can express the nucleic acid molecule or at least one strand of this nucleic acid molecule.

[0182] The present disclosure also provides a method of preparing the siRNA of the present disclosure, the method comprising culturing the aforementioned recombinant cell, or directly obtaining the siRNA using a chemical synthesis and mixing approach.

[0183] The present disclosure also provides a pharmaceutical composition comprising the siRNA duplex or corresponding siRNA conjugate described herein, and a pharmaceutically acceptable carrier.

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

[0185] The pharmaceutical composition comprising the RNAi agent of the present disclosure can be, for example, a solution with or without a buffer or a composition containing a pharmaceutically acceptable carrier. Such compositions include, for example, aqueous or crystalline compositions, liposome formulations, micelle formulations, emulsions, and gene therapy vectors.

[0186] In the methods of the present disclosure, the siRNA can be administered in a solution. A free siRNA can be administered in a non-buffered solution, for example, in physiological saline or in water. Alternatively, the free siRNA can also be administered in a suitable buffered solution. The buffered solution can include acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In a preferred embodiment, the buffered solution is phosphate buffered saline (PBS). The pH and the osmolality of the buffer containing the siRNA can be adjusted such that it is suitable for administration to a subject.

[0187] In some embodiments, the buffered solution further comprises an agent for controlling the osmolality of the solution such that the osmolality is maintained at a desired value, for example, at the physiological value of human blood plasma. Solutes that can be added to the buffered solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolizable polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In certain embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0188] The pharmaceutical compositions of the present disclosure can be administered in a dose sufficient to inhibit expression of the FXI gene. Generally, a suitable dose of the siRNA of the present disclosure is in the range of about 0.001 to about 200.0 milligrams per kilogram of body weight of the recipient per day, typically in the range of about 1 to 50 mg per kilogram of body weight per day. For example, the siRNA (e.g., siRNA conjugate) can be administered at about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg per single dose.

[0189] The pharmaceutical composition can be administered once a day, or multiple times at different intervals from 1 to 365 days, or the siRNA can be administered in two, three or more sub-doses at appropriate intervals throughout the year, or even continuously by continuous infusion or delivery using a controlled release formulation. In this case, the siRNA contained in each sub-dose must be correspondingly less so as to achieve the total daily dose. Dose units can also be compounded for delivery over several days, for example using conventional sustained release formulations that provide a sustained release of siRNA over a period of several days. Sustained release formulations are well known in the art and are particularly useful for delivering agents at a particular site, and can be used with the agents of the present disclosure. In this embodiment, the dose unit comprises a corresponding plurality of daily doses.

[0190] In other embodiments, a single dose of the pharmaceutical composition can be sustained for a long duration, such that subsequent doses are administered at intervals of no more than 3, 4, or 5 days or at intervals of no more than 1, 2, 3, or 4 weeks. In some embodiments of the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once a week. In other embodiments of the disclosure, a single dose of the pharmaceutical composition of the disclosure is administered once a month.

[0191] Those of skill in the art will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatments, the general health and / or age of the subject, and other existing diseases. In addition, treatment of a subject with a therapeutically effective dose of the composition can include a single treatment or a series of treatments. Effective doses and in vivo half-lives of the various siRNAs encompassed by the present disclosure can be estimated using conventional methods or based on in vivo testing using appropriate animal models.

[0192] The pharmaceutical compositions of the disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., through a skin patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers; intratracheal; intranasal; epidermal and transdermal, oral or parenteral. Parenteral administration includes subcutaneous, intracutaneous, intradermal, intramuscular, intraperitoneal or intravenous injection or infusion; subdermal, e.g., via implantation devices; or intracranial, e.g., intracerebral, intrathecal or intraventricular, administration.

[0193] The siRNAs for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one lipid bilayer (e.g., one lipid bilayer or multiple lipid bilayers), which has an external membrane formed of lipophilic material and an aqueous interior located inside. The lipophilic material separates the aqueous interior from the aqueous exterior, which typically does not include the siRNA composition (although in some instances, it can). Liposomes are useful for transferring and delivering active ingredients to a site of action. Because the liposome membrane is structurally similar to a biological membrane, when a liposome is administered to a tissue, the liposome bilayer fuses with the bilayer of a cell membrane. As fusion of the liposome with the cell proceeds, the internal aqueous contents, including the siRNA, are delivered into the cell, where the siRNA can specifically bind to a target RNA and can mediate RNA interference (RNAi). In some cases, the liposomes are also specifically targeted, e.g., to direct the siRNA to a particular cell type.

[0194] Liposomes containing siRNA can be prepared by a variety of methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed with the lipid components. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The siRNA preparation is then added to the micelles comprising the lipid components. The cationic groups on the lipids interact with the siRNA and condense around the siRNA to form liposomes. After condensation, the detergent is removed, e.g., by dialysis, to obtain the corresponding liposomal preparation of siRNA.

[0195] siRNAs, e.g., RNA duplexes of the disclosure, can be encapsulated in a lipid formulation (e.g., LNP or other nucleic acid-lipid particle).

[0196] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs contain a cationic lipid, a non-cationic lipid, and a lipid that prevents the particle from aggregating (e.g., a PEG-lipid conjugate). LNPs are extremely useful for synthetic applications because they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., at sites physically separate from the site of administration).

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

[0198] In some preferred embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a structural lipid, and a polymer-conjugated lipid.

[0199] In some preferred embodiments, the cationic lipid is a compound of structure (I), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C 6～15 linear alkyl; L2is C 12～25 branched alkyl. For example, YK-009 of structure (I-I), and the like (see patent CN114044741B, the entire contents of which are incorporated herein by reference, including therein the general formula and specific compounds, etc.).

[0200] (I)

[0201] (I-I)

[0202] In some preferred embodiments, the cationic lipid is a compound of structure (II), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 2~8 alkylene; G2is C 2~8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6~25 linear or branched alkyl; R2is C 6~25 linear or branched alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is HO(CH2)2- or HO(CH2)3-; L is (CH2)2- or -(CH2)3- or -(CH2)4-. For example, YK-401 of structure (II-I), YK-402 of structure (II-II), YK-407 of structure (II-III), and the like (see patent CN115784921B, the entire contents of which are incorporated herein by reference, including therein the general formula and specific compounds, etc.).

[0203] (II)

[0204] (II-I)

[0205] (II-II)

[0206] (II-III)

[0207] In some preferred embodiments, the cationic lipid is a compound of structure (III), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 1~6 alkylene; G2is C 2~8 alkylene; R1is C 6~20 linear or branched alkyl; R2is C 12~25 branched alkyl; G3is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-. For example, YK-201 of structure (III-I), YK-202 of structure (III-II), etc. (see patent CN115677518B, the entire contents of which are incorporated herein by reference, including therein the general formula and specific compounds, etc.).

[0208] (III)

[0209] (III-I)

[0210] (III-II)

[0211] In some preferred embodiments, the cationic lipid is a compound of structure (IV), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1~8 alkylene; G2is C 2~8 alkylene; R1is C 6~25 linear or branched alkyl; R2is C 12~25 linear or branched alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3is -CH3or -CH2CH3or -CH2CH2OH For example, YK-305 of the structure of Formula (IV-I), YK-310 of the structure of Formula (IV-II), and the like (see patent CN115745820B, the entire contents of which are incorporated herein by reference, including therein the general formulae and specific compounds, etc.).

[0212] (IV)

[0213] (IV-I)

[0214] (IV-II)

[0215] In some preferred embodiments, the cationic lipid is a compound of the structure of Formula (V), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G 1 and G 2 are each independently unsubstituted C6-C 10 alkylene; G 3 is unsubstituted C1-C 12 alkylene; R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is OR 5 , N, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; and R 5 is H or C1-C6 alkyl; for example, ALC0315 of the structure of Formula (V-I), and the like (see patent CN108368028B, the entire contents of which are incorporated herein by reference, including therein the general formulae and specific compounds, etc.).

[0216] (V)

[0217] (V-I)

[0218] In some preferred embodiments, the cationic lipid is a compound of the structure of Formula (VI), or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4is selected from -(CH2) n Q and -(CH2) n CHQR; Q is selected from the group consisting of -OR, -OH, -O(CH2)n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8, and heterocycle; n is 1, 2, or 3; for example, SM102 of structure (VI-I) (see patent application CN110520409A, the entire contents of which are incorporated herein by reference, including in particular the general formulae and specific compounds therein, etc.).

[0219] (VI)

[0220] (VI-I)

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

[0222] (VII)

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

[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, sterol, and derivatives thereof.

[0230] In some more preferred embodiments, the neutral lipid is selected from any one or a combination of at least two of the group consisting of 1,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-didodecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl- sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleoyl phosphatidyl ethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-stearophosphoethanolamine (SOPE), 1-stearoyl-2-oleoyl- phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof.

[0231] In some preferred embodiments, the structural lipid is selected from any one or a combination of at least two of the group consisting of: cholesterol, non-sterols, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, a-tocopherol, corticosteroids.

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

[0233] In some more preferred embodiments, the polymeric conjugated lipid is selected from any one or a combination of at least two of the group consisting of: distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristyl glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bimatiryl acetamide (ALC-0159).

[0234] Examples of pharmaceutical compositions of the present disclosure include, but are not limited to, aqueous formulations, emulsion formulations, and liposome-containing formulations. These compositions can be generated from a variety of components, examples of which include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. For example, preferred are formulations that target the liver when treating liver disorders, such as liver cancer.

[0235] Pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0236] The compositions of the present disclosure can be formulated into any of a number of possible dosage forms, such as, but not limited to, tablets, capsules, gelatin capsules, liquid syrups, soft capsules, suppositories, and enemas. The compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous, or mixed media. The aqueous suspensions can further comprise a viscosifying agent, such as, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions can also comprise a stabilizing agent.

[0237] Certain compositions of the present disclosure also incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid or its analog that is inert (i.e., not biologically active per se) but is considered a nucleic acid in vivo processes, e.g., by degrading or facilitating removal of biologically active nucleic acids from circulation, to reduce bioavailability of biologically active nucleic acids. Co-administration of a nucleic acid and a carrier compound, typically with the latter in excess, can result in a substantial reduction in the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoir, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, co-administration with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4' isothiocyanatostilbene-2,2'-disulfonic acid can reduce recovery of partially phosphorothioated dsRNA in liver tissue (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0238] A "pharmaceutical carrier" or "excipient" in contrast to a carrier compound, is a pharmaceutically acceptable solvent, suspending agent or other vehicle with which non- nucleic acid components are administered to an animal. The excipient can be liquid or solid and is selected with the planned manner of administering the particular pharmaceutical composition in mind and to provide for the most effective delivery of the nucleic acid. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or dibasic calcium phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulfate, etc.).

[0239] Pharmaceutically acceptable organic or inorganic excipients that do not destroy the pharmacological activity of the nucleic acid can also be used to formulate compositions of the present disclosure for parenteral administration. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0240] Formulations for topical administration of nucleic acids can include sterile or non-sterile aqueous solutions, non-aqueous solutions, or nucleic acid solutions in liquid or solid oil bases. These solutions also can include buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not cause toxic reactions with the nucleic acids can be used.

[0241] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0242] The present disclosure also provides methods for treating or preventing diseases and conditions that can be modulated by downregulating FXI gene expression. For example, deep vein thrombosis, venous or arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, stroke, Alzheimer's disease, thrombotic diseases associated with chronic kidney disease or end-stage renal disease.

[0243] The siRNAs of the present disclosure can be administered to a subject using any mode of administration known in the art, including, but not limited to, subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, translymphatic, trans-cerebrospinal, and any combination thereof. In preferred embodiments, the agents are administered subcutaneously.

[0244] In further embodiments, the siRNA is administered in combination with an additional therapeutic agent. The siRNA and the additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

[0245] Examples of additional therapeutic agents include agents known to treat thrombotic diseases or agents known to treat cardiovascular and cerebrovascular diseases, including, for example, anticoagulants, antiplatelet agents, and thrombolytic agents for acute myocardial infarction, pulmonary embolism, and other agents. Among them, the anticoagulants include warfarin, heparin, low molecular weight heparin (such as enoxaparin), direct oral anticoagulants (such as dabigatran, rivaroxaban, apixaban, and edoxaban); the antiplatelet agents include aspirin, clopidogrel, ticagrelor, and prasugrel; the thrombolytic agents for acute myocardial infarction and pulmonary embolism include alteplase, tenecteplase, streptokinase; and the other agents include fondaparinux and bivalirudin.

[0246] In one embodiment, the RNAi agent is administered to a patient and subsequently the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the RNAi agent and the additional therapeutic agent are administered simultaneously.

[0247] The application scenarios of "non-diagnostic or non-therapeutic purposes" described in the present disclosure include but are not limited to: as a positive control to screen other siRNA duplexes that inhibit the expression of FXI gene, or as an inhibition means to study the relationship between abnormal expression of FXI gene and diseases in the laboratory.

[0248] The nucleotide codes herein are shown in Table 1 below:

[0249] Table 1

[0250]

[0251] Examples

[0252] In the following examples, the p value of the experimental data for inter-group comparison is <0.05, and the difference is statistically significant.

[0253] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure, will become apparent to those skilled in the art from this detailed description.

[0254] The experimental techniques and experimental methods used in the present embodiment are all conventional techniques and methods, for example, the experimental methods in the following examples without specific conditions are generally performed according to the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples, if not specifically stated, can be obtained through commercial channels.

[0255] As understood by those skilled in the art, in various embodiments of the present disclosure, when the subject in the experiment is an siRNA conjugate, it includes but is not limited to the inhibition rate, IC 50 , IC 40 The experimental data and results of the siRNA conjugate can correspondingly reflect the inhibition rate, IC 50 , IC 40 of the corresponding siRNA modifier of the siRNA conjugate. There is no obstacle to understanding for those skilled in the art.

[0256] Example 1: Inhibition of FXI gene by siRNA motif

[0257] According to the human FXI mRNA sequence (NM_000128.4), 78 siRNA motifs were designed and synthesized, as shown in Table 2. Among them, APC is the basic sequence siFXIg1 of the highest active compound siFXIg1M1SP shown in the patent CN113227376B, which is used as a positive control in this study. ANC is a nonsense sequence, which is used as a negative control. After transfection into HepG2 cells by lipid nanoparticles (LNP), the inhibitory effect of each siRNA on the target gene FXI was detected by qPCR technology and ELISA, and the results are shown in Table 4.

[0258] 1.1 Synthesis of siRNA motifs

[0259] Instruments and reagents: Genesee 192 P model DNA / RNA automatic synthesizer, its solid phase carrier is a general carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (cytiva manufacturer).

[0260] Preparation method:

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

[0262] Formula 9 Formula 10

[0263] Formula 11 Formula 12

[0264] By solid-phase phosphoramidite method, nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and hydroxyl protection. Among them, when the phosphate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, oxidation, and hydroxyl protection. When the phosphorothioate linkage is used between two nucleotides, the connection of the next nucleotide monomer includes four steps of deprotection, coupling, sulfurization, and hydroxyl protection.

[0265] Specifically, the following steps are used for preparation:

[0266] Load the solid support into the designated position of the synthesizer, and obtain the corresponding product after several synthesis cycles, which include (1) deprotection, (2) coupling, (3) oxidation / sulfurization, and (4) hydroxyl protection. The cycle process and the reagents used are described as follows:

[0267] (1) Deprotection

[0268] Use 3% dichloroacetic acid toluene solution as the deprotection reagent to remove the DMT protecting group, and then use acetonitrile for washing.

[0269] (2) Coupling

[0270] Use 0.25 M 5-ethylthiotetrazole as the activating agent to couple the acetonitrile solution of each nucleotide monomer, and then use acetonitrile for washing.

[0271] (3) Oxidation / sulfurization

[0272] Oxidation: use 0.05 M iodine in pyridine / water (90 / 10) solution as the oxidizing agent for oxidation, and then use acetonitrile for washing.

[0273] Sulfurization: use 3% hydrogenated xanthate in pyridine solution as the sulfurizing agent for sulfurization, and then use acetonitrile for washing.

[0274] (4) Hydroxyl protection

[0275] Use 10% acetic anhydride tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as the hydroxyl protection reagent for hydroxyl protection, and then use acetonitrile for washing.

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

[0277] (5) Use 3% dichloroacetic acid toluene solution as the deprotection reagent to remove the DMT protecting group of the last nucleotide, and then use acetonitrile for washing.

[0278] (6) Aminolysis and purification

[0279] Transfer the reacted solid support to the reactor, add concentrated ammonia water (25-28%, mass percentage), keep aminolysis at 60°C for 12 h, then reduce the system to room temperature, filter the mixture, rinse the filter cake with a mixed solution of purified water and ethanol, combine the filtrate, pass through a chromatographic column, concentrate, freeze-dry, and obtain the 2'-O-TBDMS-protected product.

[0280] (7) De-TBDMS

[0281] To the obtained product, DMSO and triethylamine hydrofluoric acid were added, and the reaction was carried out at 60°C for 2h, then an aqueous solution of ammonium acetate was added to the reaction solution, and after shaking and mixing, anhydrous ethanol was added, and after shaking and mixing, the product was crystallized at -20°C for 8-12h. After centrifugation, the supernatant was discarded, and the precipitate was washed with anhydrous ethanol to obtain the unmodified single-stranded product.

[0282] (8) Annealing

[0283] The obtained sense strand and antisense strand of the siRNA motif were mixed at a molar ratio of 1:1, heated to 95°C and kept for 3min, and then slowly cooled to room temperature to form double-stranded siRNA motifs.

[0284] Table 2 siRNA motif

[0285]

[0286]

[0287]

[0288] 1.2 Inhibition of FXI gene by siRNA motif

[0289] 1.2.1 Experimental method

[0290] HepG2 cell strain was cultured in 10% fetal bovine serum DMEM culture medium (supplemented with 100x penicillin, 100x streptomycin 10 μL / mL) in a 37°C cell culture box containing 5% CO2.

[0291] Before transfecting the siRNA into the 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 with Opti-MEM to the appropriate concentration. For single concentration point transfection, the siRNA preparation concentration was 960 pM. 55 μL of siRNA solution diluted with Opti-MEM was added to 55 μL of transfection mixture at a ratio of 1:1 (v / v).

[0292] For the blank control group, 55 μL of prepared transfection mixture was added to 55 μL of Opti-MEM. After vortexing and mixing, it was allowed to stand at room temperature for 15 min.

[0293] The prepared transfection reagent was added to a 24-well cell culture plate (100 μL per well), and the final siRNA concentration per well was 90 pM. 500 μL of cell suspension (cell density 1.5x10 5 / mL). After mixing with cross method, put into 37℃, 5% CO2 cell incubator, culture for 48 h.

[0294] After transfection for 48 h, first extract RNA according to RNA extraction kit (RNeasy Mini Kit, QIAGEN, 74106) instruction, then reverse transcribe RNA into cDNA by using reverse transcription kit FastKing RT Kit (with gDNase) (TIANGEN, KR116-02), finally use TB Green ® Premix Ex Taq™ (Tli RNaseH Plus) kit (Takara, RR420W(L x 5)) for qPCR to quantitatively detect FXI mRNA expression level. Amplification primer in qPCR experiment is shown in Table 3. qPCR reaction program is: 95℃ heating for 30 s, then enter cycle mode, 95℃ heating for 5 s, then 60℃ heating for 34 s, for 40 cycles.

[0295] Table 3 qPCR amplification primer

[0296]

[0297] FXI mRNA expression rate (%) calculation formula is:

[0298] Expression rate = (FXI mRNA expression amount / blank control group FXI mRNA expression amount) x 100%;

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

[0300] 1.2.2 Experimental results

[0301] Transfect siRNA compound into HepG2 cells by using LNP, after 48 h, inhibition rate of each siRNA motif on FXI mRNA expression is shown in the following table.

[0302] Table 4 siRNA motif inhibition rate (%) on FXI gene

[0303]

[0304] Example 2: Inhibition effect of alternately modified siRNA on FXI gene

[0305] In order to improve inhibition rate and stability, siRNA motif in Table 2 is alternately modified by 2'-methoxy (2'-OMe) and 2'-fluoride (2'-F), and has 3', 5'-thiophosphoric acid ester bond between nucleotide at 5' and 3' end of antisense strand and 5' end of sense strand.

[0306] 2.1 Synthesis of alternately modified siRNA modifications

[0307] The rule of alternately modification in the present disclosure is that the nucleotides at the odd point positions of the sense strand and the nucleotides at the even point positions of the antisense strand are both modified with 2'-F, and the nucleotides at the other point positions are modified with 2'-OMe. In addition, there are 3', 5'-phosphorothioate bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the sense strand; there are 3', 5'-phosphorothioate bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 5' end of the antisense strand, and there are 3', 5'-phosphorothioate bonds between the 1st and 2nd nucleotides and between the 2nd and 3rd nucleotides from the 3' end of the antisense strand. The alternately modified siRNA modification designed according to this rule is represented by adding "-AL" after the original basic sequence number, as shown in Table 5.

[0308] Instrument and reagent: GenXpert 192 P model DNA / RNA automatic synthesizer, its solid phase carrier is a general carrier of cross-linked polystyrene beads, model Primer support 5G Unylinker 350 (Cytiva).

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

[0310] According to the monomer concentration of 0.15 M, the following nucleotide monomer solutions were prepared with acetonitrile: DMT-A-OMe phosphoramidite monomer (Formula 1), DMT-C-OMe phosphoramidite monomer (Formula 2), DMT-G-OMe phosphoramidite monomer (Formula 3) and DMT-U-OMe phosphoramidite monomer (Formula 4), DMT-A-F phosphoramidite monomer (Formula 5), DMT-C-F phosphoramidite monomer (Formula 6), DMT-G-F phosphoramidite monomer (Formula 7) and DMT-U-F phosphoramidite monomer (Formula 8).

[0311]

[0312]

[0313]

[0314] The nucleotide monomers are connected one by one in the order of nucleotide arrangement from 3'-5' direction by solid-phase phosphoramidite method. Each connection of a nucleotide monomer includes four steps of deprotection, coupling, oxidation or sulfurization, and hydroxyl protection. When the phosphate ester is used for connecting two nucleotides, the four steps of deprotection, coupling, oxidation, and hydroxyl protection are included when the next nucleotide monomer is connected; when the phosphorothioate is used for connecting two nucleotides, the four steps of deprotection, coupling, sulfurization, and hydroxyl protection are included when the next nucleotide monomer is connected.

[0315] (1) Deprotection

[0316] The DMT protecting group is removed by using 3% dichloroacetic acid toluene solution as a deprotection reagent, and then acetonitrile is used for cleaning.

[0317] (2) Coupling

[0318] The acetonitrile solution of each nucleotide monomer is coupled by using 0.25 M 5-ethylthiotetrazole as an activator, and then acetonitrile is used for flushing.

[0319] (3) Oxidation / Sulfurization

[0320] Oxidation: Oxidation is performed by using 0.05 M iodine in pyridine / water (90 / 10) solution as an oxidizing agent, and then acetonitrile is used for flushing.

[0321] Sulfurization: Sulfurization is performed by using 3% hydrogenated xanthate in pyridine solution as a sulfurizing agent, and then acetonitrile is used for flushing.

[0322] (4) Hydroxyl Protection

[0323] Hydroxyl protection is performed by using 10% acetic anhydride in tetrahydrofuran solution (CAP A) tetrahydrofuran / pyridine / N-methylimidazole 74 / 10 / 16 (v / v / v) (CAP B) as a hydroxyl protection reagent, and then acetonitrile is used for flushing.

[0324] The above operations are repeated, and the above steps are cyclically performed according to the set nucleotide arrangement order to obtain the sense strand product or the antisense strand product with a specific sequence arrangement.

[0325] (5) The DMT protecting group of the last nucleotide is removed by using 3% dichloroacetic acid toluene solution as a deprotection reagent, and then acetonitrile is used for cleaning.

[0326] (6) Aminolysis and purification

[0327] The solid phase carrier was transferred to a reactor, concentrated ammonia (25%-28% by mass) was added, and after ammonolysis at 60°C for 12 h, the system was brought to room temperature, and the mixture was transferred to a filter tank for filtration, the filter cake was rinsed with a mixture of purified water and ethanol, the filtrates were combined, chromatographed, concentrated, and lyophilized to obtain the 2'-OMe and 2'-F modified single-stranded product.

[0328] (7) annealing

[0329] The purified sense and antisense strands were mixed at a 1:1 molar ratio, heated to 95°C and held for 3 min, and 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, and a total of 80 siRNAs were obtained. APC-AL is the base sequence of siFXIg1M1SP (siFXIg1, see Table 2) subjected to the above fluorine-oxygen alternating modification, and is the positive control in this study. ANC-AL is the alternating modification of the nonsense sequence ANC (its base sequence is shown in Table 2), and is the negative control in this study.

[0331] 2.2 Inhibition of FXI gene by alternating modification siRNA modifiers

[0332] The inhibitory effect of the fluorine-oxygen alternating modification siRNA modifiers synthesized in Example 2.1 on the FXI gene was determined using qPCR and ELISA on HepG2 cells, and the dose-dependent effect was evaluated by a multi-concentration point transfection experiment.

[0333] 2.2.1 qPCR detection

[0334] The 80 fluorine-oxygen alternating modification sequences (base sequence shown in Table 2) were subjected to qPCR detection to evaluate their inhibitory efficiency on FXI mRNA expression, and the experimental method was referred to Example 1, 1.2.1.

[0335] The results showed that at a compound transfection concentration of 90 pM, the FXI inhibition rate of the positive control APC-AL was 51%, and the inhibition rates of A52-AL and A79-AL were 58% and 52%, respectively, which were better than that of the positive control.

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

[0337]

[0338]

[0339] 2.2.2 ELISA detection

[0340] For siRNAs with high inhibition rates detected by qPCR, in order to detect their inhibition of target gene FXI at the protein level, the supernatant of the cell culture medium of siRNA transfection for 48 h was detected by ELISA using human Factor XI ELISA kit (Thermo Fisher, EH118RB). The specific operation is as follows:

[0341] 1) Prepare the diluent and washing buffer in the kit into 1x concentration with deionized water.

[0342] 2) Dilute the standard sample by 2.5 times with ELISA diluent to prepare the standard sample.

[0343] 3) Add 100 μL of 5-fold diluted sample, standard sample and ELISA diluent to the corresponding wells of the antibody pre-coated 96-well plate, 2 replicate wells for each sample, cover the lid, and incubate at room temperature for 2.5 h.

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

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

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

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

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

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

[0350] 10) Discard the reaction solution, wash 4 times with washing buffer, 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) Read the absorbance at 450 nm using a microplate reader.

[0354] The ELISA data was processed to normalize to the transfection reagent control group (relative expression to the transfection reagent control group = protein expression of each sample FXI / protein expression of the transfection reagent control group sample FXI).

[0355] The results show (Table 6) that at a 90 pM compound transfection concentration, the fluoroxy alternately modified siRNA APC-AL has an FXI inhibition rate of 54%, and the A3-AL and AL52-AL have inhibition rates of 57% and 60%, respectively, which are superior to the positive control.

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

[0357]

[0358] 2.2.3 IC 50 determination

[0359] The 16 sequences that showed high inhibition rates in qPCR and ELISA experiments under single concentration point (90 pM) transfection conditions were subjected to multi-concentration point transfection to fit IC 50 to evaluate their dose-dependent effects.

[0360] The experimental method is as in 1.2.1, except that 8 concentration points were transfection, and the siRNA transfection concentrations were 10.0000, 2.5000, 0.6250, 0.1563, 0.0391, 0.0098, 0.0024 and 0.0006 nM. After calculating the inhibition rate of each concentration point, the dose-effect relationship curve of the compound was fitted using GraphPad Prism software (four parameter logistic equations), and the IC 50 of the compound was calculated.

[0361] The results show (Table 7) that the IC 50 values of A52-AL, A64-AL, A74-AL, A3-AL, A7-AL, A50-AL and A75-AL are superior to the positive control APC-AL.

[0362] Table 7 IC 50 determination

[0363]

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

[0365] Example 3: Inhibition rate comparison of siRNA motifs of the present disclosure and similar sequences in prior art

[0366] The 8 siRNA motifs of the present disclosure, i.e. A3, A7, A50, A52, A64, A74, A75 and A79, showed better in vitro pharmacodynamic activity in Example 2. Among them, 6 siRNA motifs A3, A50, A64, A74, A75 and A79 are similar to the sequences disclosed in prior art. This example compares the inhibition rate of FXI gene at the in vitro cell level for these 6 sequences.

[0367] 3.1 Synthesis of siRNA motifs

[0368] The siRNA motifs of the prior art in Table 8 were synthesized using the synthesis method of 1.1 in Example 1.

[0369] Table 8 Similar sequence information in prior art

[0370]

[0371] 3.2 Comparison of inhibition of FXI gene by siRNA motifs

[0372] The experimental method refers to 1.2.1 in Example 1, and the siRNA transfection concentration is 90 pM.

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

[0374] Table 9 In vitro pharmacodynamic comparison with similar sequences disclosed in prior art

[0375]

[0376] From the above table, it can be seen that the inhibition rate comparison of 6 basic sequences (i.e. A3, A50, A64, A74, A75 and A79) of the present disclosure and similar motifs of prior art is carried out. The results show that under the same concentration conditions, the inhibition rate of the above 6 motifs of the present disclosure on the expression of FXI mRNA in HepG2 cells is better than that of similar sequences in 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, and the siRNA duplex comprises any one or a combination of at least two of the following oligonucleotide duplexes composed of sense strand and antisense strand pairings: (1) The sequence of the positive chain is shown in SEQ ID NO: 2; and the sequence of the negative chain is shown in SEQ ID NO: 82; (2) The sequence of the positive chain is shown in SEQ ID NO: 62; and the sequence of the negative chain is shown in SEQ ID NO:

142.

2. 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, and the siRNA duplex comprises any one or a combination of at least two of the following oligonucleotide duplexes composed of sense strand and antisense strand pairings: (1) The sense strand is a modified fragment of the sequence shown in SEQ ID NO: 2, and the antisense strand is a modified fragment of the sequence shown in SEQ ID NO: 82; wherein all nucleotide modifications on the sense strand and the antisense strand are 2' position modifications of the nucleotide ribose, and the 2' position modifications of the nucleotide ribose are as follows: the odd positions of the sense strand are all 2'-fluorinated, and the even positions are all 2'-methoxyinated; and the odd positions of the antisense strand are all 2'-methoxyinated, and the even positions are all 2'-fluorinated; 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 5' end of the antisense strand and between the second and third nucleotides at the 3' end of the antisense strand. (2) The sense chain is a modified fragment of the sequence shown in SEQ ID NO: 62, and the antisense chain is a modified fragment of the sequence shown in SEQ ID NO:

62. The modified fragment of the sequence shown in 142; wherein all nucleotide modifications on the sense and antisense strands are 2'-position modifications of the ribose nucleotides, and the 2'-position modifications of the ribose nucleotides are as follows: odd-numbered positions on the sense strand are all 2'-fluorinated, and even-numbered positions are all 2'-methoxyinated; and odd-numbered positions on the antisense strand are all 2'-methoxyinated, and even-numbered positions are all 2'-fluorinated; 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 5' end of the antisense strand and between the second and third nucleotides at the 3' end of the antisense strand.

3. A conjugate of siRNA, characterized in that, The conjugate includes the siRNA duplex as described in claim 1 or 2, and the conjugate group attached thereto.

4. 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 claim 1 or 2, 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 claim 1 or 2, and a nuclease.

5. The nucleic acid-protein complex according to claim 4, characterized in that, The nuclease is an AGO protein.

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

7. The use of the siRNA double strand as described in claim 1 or 2, the conjugate as described in claim 3, the nucleic acid protein complex as described in claim 4 or 5, or the pharmaceutical composition as described in claim 6 in the preparation of a medicament for the prevention or treatment of diseases related to FXI gene expression; wherein the diseases related to FXI gene expression are thrombotic diseases.

8. The use of the siRNA duplex as described in claim 1 or 2, the conjugate as described in claim 3, the nucleic acid-protein complex as described in claim 4 or 5, or the pharmaceutical composition as described in claim 6 in the preparation of an anticoagulant for diseases related to FXI gene expression; wherein the diseases related to FXI gene expression are thrombotic diseases.

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