FXI-targeted RNAi agent, and preparation method and application thereof

Abstract

The invention provides an FXI-targeted RNAi agent, a preparation method and application thereof, and further relates to application of the RNAi agent and a pharmaceutical composition containing the RNAi agent to prevention or treatment of FXI-related diseases, preferably thrombus-related diseases.

Description

RNAi agent targeting FXI, preparation method and use thereof

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an RNAi agent targeting FXI, a preparation method and use thereof, and further relates to the use of the RNAi agent and a pharmaceutical composition containing the RNAi agent for preventing or treating diseases related to FXI, preferably thrombus-related diseases.

[0002] Plasma coagulation factor XI (Factor XI, hereinafter referred to as FXI) is a key component of the contact activation pathway, which helps to produce thrombin, and thrombin is an important component involved in the formation of fibrin and the protection of fibrinolysis. High FXI level is a risk factor for venous thrombosis. By inhibiting the expression of the FXI gene, thrombotic diseases, particularly venous thrombosis and ischemic stroke, can be prevented and treated at the cellular level.

[0003] Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating diseases. The related prior art currently includes CN102245186A, CN113227376A, WO2022028457A1, WO2022221441A2, all of which are in the research stage, no products have been marketed, and the specific application still has unmet needs.

[0004]

[0005] In view of the problems existing in the prior art, the present application aims to provide an RNAi agent targeting FXI, a preparation method and use thereof, and further relates to the use of the RNAi agent and a pharmaceutical composition containing the RNAi agent for preventing or treating diseases related to FXI, preferably thrombus-related diseases.

[0006] In the present application, FXI mRNA refers to mRNA with the sequence shown in Genbank registration number NM_000128.4. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to the gene transcribing the above-mentioned FXI mRNA, and the term "target mRNA" refers to the above-mentioned FXI mRNA.

[0007] Specifically, the present application provides an RNAi agent targeting FXI gene, which comprises a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex region, wherein the sense strand and the antisense strand are selected from one or more of the combinations, preferably from the RNAi agents of Table 1.

[0008] Further, the sense strand and / or the antisense strand independently comprises one or more modified nucleotides.

[0009] Further, the one or more modified nucleotides can be independently selected from the group consisting of any one of a 5'-phosphorothioate based nucleotide, a 5'-methylated cytosine nucleotide, a 5'-methylphosphonate nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-2-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, an alkyl nucleotide, a 5'-C-methylphosphonate based nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a 2'-fluoro modified nucleotide, a 3'-nitrogen substituted modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a vinyl phosphonate deoxyribonucleotide, a phosphorothioate nucleotide, a phosphorodithioate nucleotide, a locked nucleic acid (LNA), an abasic nucleotide, a deoxythymidine, an inverted deoxythymidine, a 2'-amino modified nucleotide, a morpholino oligonucleotide (PMO), a polypeptide nucleotide, a phosphoramidate, or a non-natural base nucleotide.

[0010] Further, the modified nucleotide sequence is selected from the RNAi agents of Table 4.

[0011] Further, the RNAi agent is linked to a targeting ligand.

[0012] Further, the targeting ligand is linked to the 3' or 5' end of the sense strand.

[0013] Further, the targeting ligand comprises an N-acetyl-galactosamine (GalNac) moiety.

[0014] Further, the targeting ligand can be selected from the following structures:

[0015] Further, the RNAi agent linked to a targeting ligand sequence is selected from the RNAi agents of Table 8.

[0016] Further, the present application also provides a pharmaceutical composition comprising the RNAi agent of any one of the preceding, and one or more than one pharmaceutically acceptable excipient and / or carrier.

[0017] Further, the present application also provides the use of the RNAi agent or the pharmaceutical composition of any one of the preceding in the manufacture of a medicament for the prevention or treatment of a disease associated with FXI, preferably a thrombosis related disease.

[0018] In some embodiments, the pharmaceutical compositions of the present application can be administered in a variety of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be made by any of the means known in the art, such as, but not limited to, topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizers, intratracheal, intranasal), dermal, transdermal, oral or parenteral. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous (via an implanted device), intracranial, intrahepatic, intrathecal and intraventricular administration. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[0019] In some embodiments, the level of gene expression and / or mRNA level of FXI in a subject administered the FXI RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject prior to administration of the FXI RNAi agent or a subject not receiving the FXI RNAi agent. The level of gene expression and / or mRNA level in a subject can be reduced in a cell, cell population, and / or tissue of the subject. In some embodiments, the level of protein of FXI in a subject administered the FXI RNAi agent is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to the subject prior to administration of the FXI RNAi agent or a subject not receiving the FXI RNAi agent. The level of protein in a subject can be reduced in a cell, cell population, tissue, blood, and / or other fluid of the subject. Reduction in the level of gene expression, mRNA, or protein can be assessed by any method known in the art. Reduction or decrease in the level of FXI mRNA and / or protein is collectively referred to herein as reduction or decrease of FXI, or inhibition or reduction of expression of FXI.

[0020] The present application also provides a method of making the nucleic acid of any one of the preceding claims, wherein the sense strand and the antisense strand comprised by the nucleic acid can be conveniently and routinely made by the known techniques of solid phase synthesis. Any other method known in the art for such synthesis, such as liquid phase synthesis or fermentation, can additionally or alternatively be used. The use of similar techniques to make other oligonucleotides (such as phosphorothioate and alkylated derivatives) is known.

[0021] The term "comprising" as used herein is used in the sense of "including", and the may be used interchangeably therewith. Unless explicitly stated, the context will not dictate otherwise.

[0022] The term "silence," "reduce," "inhibit," "down-regulate," or "knock down gene expression" when referring to a FXI gene means that the expression of the gene (as measured by the level of RNA transcribed from the gene in a cell, cell population, or tissue in which the FXI gene is transcribed or the level of a polypeptide, protein, or protein subunit translated from the mRNA) is reduced when the cell, cell population, or tissue is treated with the FXI RNAi agent compared to the same cell, cell population, or tissue prior to administration of the FXI RNAi agent.

[0023] The term "antisense strand" generally refers to the strand of an RNAi agent that includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" generally 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, the mismatches can be internal or at the end regions of the molecule. Generally, the most tolerated mismatches are at the end regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' end and / or the 3' end.

[0024] The term "sense strand" generally refers to the strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as defined herein. The "sense" strand is sometimes referred to as the "plus" strand, the "passenger" strand, or the "anti-guide" strand. By virtue of their sequences, the antisense strand targets the desired mRNA while the sense strand targets a different target. Thus, if the antisense strand is incorporated into RISC, the correct target is targeted. Incorporation of the sense strand can result in off-target effects. These off-target effects can be limited by using modifications on the sense strand or using a 5' end cap.

[0025] In the present application, "complementary" has the meaning well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. The purine base adenine (A) always pairs with the pyrimidine base uracil (U); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair includes one purine and one pyrimidine. When the adenine on one strand always pairs with the uracil on the other strand, and the guanine always pairs with the cytosine, the two strands are considered to be complementary to each other, and the sequence of one strand can be inferred from the sequence of its complementary strand.

[0026] The term "complementary" when used in reference to a first nucleotide sequence (such as the sense strand of an RNAi agent) in relation to a second nucleotide sequence (such as the antisense strand of an RNAi agent) means the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base pair hydrogen bonds) and form a duplex or double helix structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetics, as long as the above requirements regarding their hybridization ability are fulfilled. "Complementary" does not necessarily have nucleobase complementarity at every nucleoside. Rather, some mismatches can be tolerated.

[0027] The term "fully complementary" generally means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The contiguous sequence can comprise all or a portion of the first or second nucleotide sequence. As used herein, "partially complementary" generally means that in a pair of hybridizing nucleobase sequences, at least about 70% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. As used herein, "substantially complementary" generally means that in a pair of hybridizing nucleobase sequences, at least about 90% of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. The terms "complementary," "fully complementary," and "substantially complementary" as used herein can be used in terms of base pairing between the sense and antisense strands of an RNAi agent or between the antisense strand of an RNAi agent and the sequence of an FXI mRNA. Sequence identity or complementarity is independent of modification. For purposes of determining identity or complementarity, for example, a and Af are complementary to U (or T) and identical to A.

[0028] In the present invention, the modified nucleotides include, but are not limited to, 5'-phosphorothioate nucleotides, 5'-methylated cytosine nucleotides, 5'-methylphosphonate nucleotides, 2'-O-methyl-modified nucleotides, inverted 2'-O-methyl nucleotides, 3'-O-methyl nucleotides, 2'-O-2-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, alkyl nucleotides, 5'-C-methylphosphonate nucleotides, 2'-F-arabinonucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNA), 2'-fluoro-modified nucleotides, 3'-nitrogen-substituted modified nucleotides, 2'-deoxy-2'-fluoro-modified nucleotides, 5'-methyl-2'-fluoro nucleotides, 2'-deoxy-modified nucleotides, vinylphosphonate deoxyribonucleotides, phosphorothioate nucleotides, phosphorodithioate nucleotides, locked nucleic acids (LNA), abasic nucleotides, abasic ribose (Ab), inverted deoxyribonucleotides (3'-3' linked nucleotides), phosphoethylene deoxyribonucleotides, inverted abasic nucleotides, deoxythymidine, inverted deoxythymidine, 2'-amino-modified nucleotides, morpholino oligonucleotides (PMO), polypeptide nucleotides, phosphoramidate, or non-natural base nucleotides.

[0029] wherein the alkyl-modified nucleotides, such as methyl nucleotides, ethyl nucleotides,

[0030] methoxy-modified nucleotides, such as, methoxyethyl nucleotides, such as, fluoro nucleotides, such as, 5'-C-methylphosphonate nucleotides, such as

[0031] vinylphosphonate deoxyribonucleotides, such as, phosphorothioate nucleotides, such as,

[0032] wherein Base represents a base, R represents an alkyl group, Me represents a methyl group, and Et represents an ethyl group.

[0033] The term "locked nucleic acid" is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an extra bridge connecting the 2' carbon and the 4' carbon. This structure effectively "locks" the ribose in a 3 '-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al. (2005) Nucleic Acids Research 33(1): 439-447; Mook, OR. et al. (2007) Mol Cancer Ther 6(3): 833-843; Grunweller, A. et al. (2003) Nucleic Acids Research 31(12): 3185-3193).

[0034] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are incorporated herein by reference.

[0035] In certain embodiments, sugar surrogates include rings having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0036] The term "morpholino" means a sugar surrogate having the following formula:

[0037] In certain embodiments, morpholino groups can be modified, e.g., by adding or altering various substituents from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholino groups."

[0038] In the present disclosure, capital letters C, G, U, A represent the base composition of a nucleotide, unless otherwise specified. Lowercase letters c, g, u, a represent the corresponding nucleotide represented by the capital letter is modified by methoxy; underscore represents the nucleotide represented by the capital letter is modified by fluorination; the dash "·" represents the two nucleotide residues adjacent to the left and right of the dash "·" are connected by phosphorothioate group; VP represents the nucleotide to the right of the letter VP is (E)-vinyl phosphate modified nucleotide. For example, "a·g" represents the a and g residues are connected by phosphorothioate group.

[0039] It should be emphasized that the "modification" of the nucleotide described in the present disclosure includes but is not limited to the above examples, and the nucleotide can also be replaced by other nucleotides, such as (S)-glycerol nucleic acid, etc.

[0040] The term "homologous" or "homology" generally refers to the number of nucleotides of a subject nucleic acid sequence that have matched the same nucleotides of a reference nucleic acid sequence, typically determined by a sequence analysis program (e.g., Karlin and Altschul, 1990, PNAS 87:2264-2268; Karlin and Altschul, 1993, PNAS 90:5873-5877), or by visual inspection. As used herein, the term "complete homology" or "completely homologous" generally refers to complete (100%) homology or "identity" between a reference sequence and a subject nucleic acid sequence. As used herein, the term "substantially homologous" or "substantial homology" generally refers to a subject sequence sharing at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) of the homologous nucleotides at the same nucleotide positions in a reference sequence.

[0041] The terms "induce", "inhibit", "enhance", "elevate", "increase", "decrease", "reduce", and the like generally indicate a quantitative difference between two states. For example, "an amount effective to inhibit the activity or expression of FXI" means that the level of FXI activity or expression in a treated sample will be lower than the level of FXI activity or expression in an untreated sample. The terms apply, for example, to expression levels and activity levels. The terms "decrease" and "reduce" are used interchangeably and generally mean any change that is less than the original. "Decrease" and "reduce" are relative terms, requiring a comparison between before and after a measurement. "Decrease" and "reduce" include complete depletion.

[0042] The term "at least" includes the number next to the term "at least" and all subsequent numbers or integers logically included therein, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 19 of the 21 nucleotides in a nucleic acid molecule" means that 19, 20, or 21 nucleotides have the indicated property. When "at least" precedes a series of numbers or a range, it is understood that "at least" can modify each number in the series or range.

[0043] The term "decrease" refers to an overall decrease in the expression level / amount of a gene, gene product, e.g., protein, or biomarker in a first sample compared to the expression level / amount of the corresponding gene, gene product, e.g., protein, or biomarker in a second sample, by about 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%, as detected by standard methods known in the art, such as those described herein. In certain embodiments, the term "decrease" refers to a decrease in the expression level / amount of a gene or biomarker in a first sample, wherein the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold of the expression level / amount of the corresponding gene or biomarker in a second sample. In certain embodiments, the first sample is a sample obtained from a subject, and the second sample is a reference sample.

[0044] The term "expression" generally refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of a 5'-cap), and translation.

[0045] The term "pharmaceutically acceptable" generally refers to a substance that is not toxic to the extent that it interferes with the effectiveness of an active ingredient's biological activity. Such formulations can generally contain a salt, excipient, buffer, preservative, compatible carrier, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations can also generally contain compatible solid or liquid fillers, diluents, or encapsulating material that are suitable for administration to humans. When used in medicine, salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can conveniently be used to prepare pharmaceutically acceptable salts, and are not excluded from the scope of the application. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts derived from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.

[0046] Pharmaceutical compositions of the present disclosure include those suitable for oral, nasal, topical, buccal, sublingual, rectal, and / or parenteral administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of a compound that produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of the dosage, preferably from about 5% to about 70%, most preferably from about 10% to about 30% by weight of the active ingredient, in terms of percentages.

[0047] The term "preventing and / or treating" includes not only the prevention and / or treatment of a disease, but also generally includes preventing the onset of a disease, slowing or reversing the progression of a disease, preventing or slowing the onset of one or more symptoms associated with a disease, reducing and / or alleviating one or more symptoms associated with a disease, reducing the severity and / or duration of a disease and / or any symptoms associated therewith and / or preventing further increases in the severity of a disease and / or any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by a disease, and generally any pharmacological action that is beneficial to the patient being treated. The nucleic acid or pharmaceutical composition of the present application forms a viable therapeutic agent without requiring the complete cure or eradication of any symptom or manifestation of a disease. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but need not eliminate every manifestation of a disease to be considered a useful therapeutic agent. Similarly, a therapeutic agent prophylactically administered forms a viable prophylactic agent without being completely effective in preventing the onset of a disorder. It is sufficient to simply reduce the impact of a disease in a subject (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another therapy, or by producing another beneficial effect), or to reduce the likelihood of a disease from occurring or worsening.

[0048] The term "disease" or "disorder" can be used interchangeably and generally refers to any deviation from the normal state of a subject, for example, any change in the state of the body or of some of its organs, impairs or interferes with the performance of the functions, and / or causes symptoms such as discomfort, dysfunction, pain, or even death in a person affected or exposed to it. A disease or disorder can also be referred to as a distemper, an ailing, an ailment, a malady, a disorder, a sickness, an illness, a complaint, an inderdisposion, or an affectation.

[0049] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present application, prepared from a compound of the present application having specific substituents discovered herein with a pharmaceutically acceptable acid or base.

[0050] The term "pharmaceutically acceptable carrier" means any formulation carrier or medium that does not interfere with the effectiveness of the active substance of the application, that is not toxic to the host or patient, and that is otherwise suitable to be used in vivo. Representative carriers include water, oil, vegetable and mineral, cream bases, lotion bases, ointment bases, and the like. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, and the like. Their formulation is well known to those skilled in the art of cosmetics or topical pharmaceuticals. Further information on carriers can be found in Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0051] The term "excipient" generally refers to carriers, diluents, and / or media required for formulating an effective pharmaceutical composition. Among others, excipients include (but are not limited to) absorption enhancers, anti-adherents, antifoaming agents, antioxidants, binders, buffering agents, carriers, coating agents, coloring agents, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, effervescent agents, fillers, flavoring agents, glidants, humectants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavorings, and fragrances.

[0052] The term "effective amount" or "therapeutically effective amount" with respect to a pharmaceutical or pharmacological agent means a sufficient amount of the agent to achieve the intended effect without being toxic to the recipient. For oral dosage forms of the application, an "effective amount" of one active substance in a composition means the amount required to achieve the intended effect in conjunction with another active substance in the composition. The determination of an effective amount is dependent on the age and general condition of the recipient, as well as the particular active substance, and an appropriate effective amount for a given case can be determined by one of ordinary skill in the art based on routine testing.

[0053] The term "active ingredient", "therapeutic agent", "active substance", or "active agent" means a chemical entity that is effective in treating a target disorder, disease, or condition.

[0054] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0055] The compounds of the present application can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by a combination of the specific embodiments set forth below with other chemical synthetic methods well known in the art, and equivalents thereof as appreciated by those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present application.

[0056] Example 1 Synthesis of RNAi agents

[0057] RNAi agents were prepared using OligoMaker ApS 192 RNA synthesizer (Denmark), and the specific synthesis route can refer to the patent document CN113227376A. The designed RNAi agent sequences are shown in Table 1 below.

[0058] Table 1 RNAi agent sequences

[0059] Example 2 In vitro inhibitory activity test of FXI RNA agent in Hep3B cells

[0060] Hep3B cells were cultured in MEM (Gibco-11095080) medium containing 10% fetal bovine serum (ExCell Bio-FSP500), 1% Non Essential Amino Acids (Gibco-11140050), 1mM Sodium Pyruvate (Gibco-11360070) and 1% penicillin-streptomycin (HyClone-SV30010). The siRNA diluted with RNase-free water and RNAi MAX (Invitrogen-13778075) transfection reagent were added to the Hep3B cell suspension, and plated in a 96-well plate at a cell density of 2x10 5 / ml, so that the final concentration of siRNA was 10 and 0.1nM. After 48 hours of culture, total RNA was extracted from the cells (QIAGEN-74182), reverse transcribed and subjected to qPCR to determine the expression level of FXI mRNA by SYBR Green method.

[0061] The expression level of the target gene mRNA of each sample was calculated by ΔΔCT relative quantification method. The relative expression level of the target gene was calculated using 2 -ΔΔCTThe relative expression value of FXI mRNA was calculated by the following equation. -ΔΔCT The relative expression value of FXI mRNA was calculated by the following equation.

[0062] The inhibition rate of FXI gene mediated by RNA agent was calculated by the following equation.

[0063] FXI Inhibition % = (1 - value of sample / Ave. value of RNAiMAX Control) x 100

[0064] The inhibition rate of FXI gene mediated by RNA agent was calculated by the following equation.

[0065] Table 2

[0066] Example 3 In vitro inhibition activity test of FXI RNA agent in SEAP reporter gene system

[0067] The full length of human FXI gene (including 5'UTR and 3'UTR sequence) was cloned into pDRIVE5s-SV40hALB vector to construct a plasmid containing the full length sequence of human FXI and secreted alkaline phosphatase (SEAP) 3'UTR. HuH7 cells were cultured in DMEM (Gibco-10313021) medium containing 10% fetal bovine serum (Gibco-10099-141), 1% glutamine (Gibco-35050061), 1% non-essential amino acids (Gibco-11140050), 1% penicillin-streptomycin (HyClone-SV30010). FXI-pDRIVE5s-SV40hALB plasmid diluted with Opti-MEM (Gibico-11058021), RNA agent and Lipo2000 (Invitrogen-11668019) transfection reagent were added to the suspension of HuH7 cells, and plated in a 96-well plate at a cell density of 1*10^4 / ml, so that the final concentration of RNA agent was 1 nM and 0.05 nM. After 48 hours of culture, the SEAP reporter gene detection system was processed according to the test steps, and after incubation, the luminescence value was detected by enzyme label instrument (BMG, PHERAstar). TM The SEAP reporter gene detection system was processed according to the test steps, and after incubation, the luminescence value was detected by enzyme label instrument (BMG, PHERAstar).

[0068] The inhibition rate of the FXI gene mediated by the RNAi agent is calculated according to the following formula.

[0069] FXI gene inhibition rate (%) = (1-experimental group luminescence value / control group average luminescence value) x 100

[0070] The test results are as follows:

[0071] Table 3

[0072] From the experimental results of Examples 2 and 3, it can be seen that the RNA agent of the present application has good inhibitory activity on FXI mRNA.

[0073] Synthesis of modified sequences of siRNA analogs in Example 4

[0074] An OligoMakerApS 192 RNA synthesizer (Denmark) is used to prepare siRNA analogs containing modifications, wherein the specific synthesis route can refer to patent document CN113227376A, and the sequences S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17, S23, S24, S25, S26, S27, S28, S29, S44-S54 modified siRNA analog sequences are shown in Table 4.

[0075] Table 4 is a modification sequence table

[0076] wherein A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Am-s = 2'-O-methyladenosine-3'-phosphorothioate; Um-s = 2'-O-methyluridine-3'-phosphorothioate; Cm-s = 2'-O-methylcytidine-3'-phosphorothioate; Gm-s = 2'-O-methylguanosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Af-s = 2'-fluoroadenosine-3'-phosphorothioate; Uf-s = 2'-fluorouridine-3'-phosphorothioate; Cf-s = 2'-fluorocytidine-3'-phosphorothioate; Gf-s = 2'-fluoroguanosine-3'-phosphorothioate; dA = 2'-deoxyadenosine-3'-phosphate; dC = 2'-deoxycytidine-3'-phosphate; dG = 2'-deoxyguanosine-3'-phosphate; dT = 2'-deoxythymidine-3'-phosphate; m = 2'-O-methyl; f = 2'-fluoro; s = phosphorothioate linkage.

[0077] Example 5 In vitro activity of modified sequences of siRNA analogs

[0078] The in vitro activity of the sequences was tested using the method of Example 2, wherein the activity of sequences SI-SI 6 is shown in Table 5.

[0079] Table 5, FXI inhibition rate (10 nm) of modified sequences

[0080] Example 6 In vitro activity of modified sequences of siRNA analogs

[0081] The in vitro activity of the sequences was tested using the method of Example 3, wherein the activity of sequences S24, S25, S26, S27, S28 is shown in Table 6, respectively; and the activity of sequences S44-S54 is shown in Table 7.

[0082] Table 6, FXI inhibition rate (1 nm) of modified sequences

[0083] Table 7, FXI inhibition rate (1 nm and 0.05 nm) of modified sequences

[0084] wherein A = adenosine-3'-phosphate; U = uridine-3'-phosphate; C = cytidine-3'-phosphate; G = guanosine-3'-phosphate; Am = 2'-O-methyladenosine-3'-phosphate; Um = 2'-O-methyluridine-3'-phosphate; Cm = 2'-O-methylcytidine-3'-phosphate; Gm = 2'-O-methylguanosine-3'-phosphate; Am-s = 2'-O-methyladenosine-3'-phosphorothioate; Um-s = 2'-O-methyluridine-3'-phosphorothioate; Cm-s = 2'-O-methylcytidine-3'-phosphorothioate; Gm-s = 2'-O-methylguanosine-3'-phosphorothioate; Af = 2'-fluoroadenosine-3'-phosphate; Uf = 2'-fluorouridine-3'-phosphate; Cf = 2'-fluorocytidine-3'-phosphate; Gf = 2'-fluoroguanosine-3'-phosphate; Af-s = 2'-fluoroadenosine-3'-phosphorothioate; Uf-s = 2'-fluorouridine-3'-phosphorothioate; Cf-s = 2'-fluorocytidine-3'-phosphorothioate; Gf-s = 2'-fluoroguanosine-3'-phosphorothioate; dA = 2'-deoxyadenosine-3'-phosphate; dC = 2'-deoxycytidine-3'-phosphate; dG = 2'-deoxyguanosine-3'-phosphate; dT = 2'-deoxythymidine-3'-phosphate; m = 2'-O-methyl; f = 2'-fluoro; s = phosphorothioate linkage.

[0085] Example 7 In vivo FXI silencing effects of siRNAs in hFXI expressing mice

[0086] The plasmid carrying human FXI and capable of expressing SEAP protein was introduced into Balb / c mice by high-pressure injection through the tail vein to construct a mouse model expressing human FXI (hFXI). The specific operation is as follows: 6-8 week old male Balb / c mice were selected, and 10 μg of plasmid carrying human FXI and SEAP protein 3'UTR full-length sequence was injected through the tail vein by high-pressure injection, and the injection volume was 10% of the animal body weight. Two weeks later, the siRNA knockdown ability of the target gene was tested. One day before administration (day 0), all mice were taken blood from the eye orbit into serum separation tubes, and the blood was allowed to clot at ambient temperature for 20 minutes. The tubes were centrifuged at 8,000 x g for 3 minutes to separate the serum and stored at 4°C. The SEAP expression level in the serum was detected by Phospha-LightTM SEAP reporter gene assay system (Invitrogen), and the mice were grouped according to the average SEAP level, with 6 mice in each group. The siRNA drug was administered by subcutaneous injection, and the injection site was the loose skin in the neck and shoulder area, with a dose of 3 mg / kg, and the injection volume for each mouse was 200 μL / 20 g. The day of administration was day 1, and the serum was collected from all mice by taking blood from the eye orbit on day 7, 14, 21 and 28 after administration for detecting the expression level of SEAP protein in the serum. In data processing, first, the SEAP level of each animal at a time point was divided by the expression level of the animal before treatment (day 0) to determine the expression "normalized to pre-treatment" ratio. Then, by dividing the "normalized to pre-treatment" ratio of a single animal (Ratio administration) by the average "normalized to pre-treatment" ratio of all mice in the vehicle control group (Average Ratio vehicle), the expression at a specific time point was normalized to the control group, and the inhibition rate was calculated, so the inhibition rate = (1-Ratio administration / Average Ratio vehicle)*100.

[0087] The modified sequence of GalNAc L96 and its effect data are shown in Table 8 and Table 9, respectively.

[0088] Table 8

[0089] The test results are as follows:

[0090] Table 9

[0091] The structure of L96 is as follows:

[0092] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

An RNAi agent targeting FXI, characterized in that, The RNAi agent comprises a sense strand and an antisense strand, which form a double-stranded region, wherein the sense strand and antisense strand are selected from the RNAi agents in Table 1. The RNAi agent according to claim 1 is characterized in that, The sense strand and / or antisense strand independently contain one or more modified nucleotides. The RNAi agent according to claim 2 is characterized in that, The modified nucleotide sequence is selected from the RNAi agents in Table 4. The RNAi agent according to any one of claims 1-3 is characterized in that, The RNAi agent is linked to a target ligand. The RNAi agent according to claim 4 is characterized in that, The targeting ligand is attached to the 3' or 5' end of the sense chain. The RNAi agent according to claim 4 is characterized in that, The targeting ligand contains the N-acetyl-galactosamine (GalNAc) moiety. The RNAi agent according to claim 4 is characterized in that, The RNAi agent and the target ligand linking sequence are selected from the RNAi agents in Table 8. A pharmaceutical composition, characterized in that, It comprises the RNAi agent according to any one of claims 1-7, and one or more pharmaceutically acceptable excipients and / or carriers. Use of the RNAi agent according to any one of claims 1-7 or the pharmaceutical composition according to claim 8 in the preparation for the prevention or treatment of diseases related to FXI. The use according to claim 9 is characterized in that, The FXI-related diseases are selected from thrombosis-related diseases.