Nucleic acid, pharmaceutical composition containing the nucleic acid and siRNA conjugate, preparation method and use thereof

Abstract

The present disclosure provides an siRNA that inhibits the expression of plasma kallikrein (PKK) gene, a pharmaceutical composition containing the siRNA, and an siRNA conjugate. Each nucleotide in the siRNA is independently a modified or unmodified nucleotide, and the siRNA contains a sense strand and an antisense strand, the sense strand contains a nucleotide sequence I, and the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 1 in length, and no more than 3 nucleotide differences, and the antisense strand contains a nucleotide sequence II, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO: 2 in length, and no more than 3 nucleotide differences. The siRNA, pharmaceutical composition, and siRNA conjugate provided by the present disclosure can effectively treat and / or prevent pathological conditions or diseases caused by the expression of plasma kallikrein gene.

Description

Technical Field

[0001] The present disclosure relates to a nucleic acid capable of inhibiting the expression of plasma kallikrein (PKK) gene and a pharmaceutical composition containing the nucleic acid and a siRNA conjugate. The present disclosure also relates to the preparation method and use of the nucleic acid, pharmaceutical composition and siRNA conjugate. Background Art

[0002] Bradykinin (BK) is a major regulator of vascular permeability. Excessive BK increases vascular leakage, thereby exacerbating inflammation. Research has shown that genetic defects in C1-esterase inhibitor (C1-INH), the main natural inhibitor of BK, can lead to hereditary angioedema (HAE). Patients with the rare disease HAE often suffer from acute attacks of painful edema caused by unknown triggers, and attacks located in the throat can be life-threatening.

[0003] Prekallikrein (PKK) is a precursor of plasma kallikrein (PK). PKK is converted to PK under the activation of factor XIIa (FXIIa). PK cleaves high molecular weight kininogen to release bradykinin into the blood vessels. Therefore, by inhibiting the expression of the PKK gene, BK excess can be suppressed at the cellular level, thereby preventing and treating diseases or symptoms such as inflammation caused by BK excess, particularly hereditary angioedema. 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.

[0004] One of the keys to developing siRNA drugs to inhibit PKK gene expression and treat hereditary angioedema is to find suitable siRNA and its modification, as well as an effective delivery system. Summary of the Invention

[0005] The inventors of the present disclosure unexpectedly discovered that the following siRNA and its modified sequence provided by the present disclosure can specifically inhibit the expression of the PKK gene, and the pharmaceutical composition or siRNA conjugate containing the siRNA can specifically target the liver, thereby inhibiting the expression of the PKK gene in the liver, achieving the treatment or prevention of inflammatory diseases, especially hereditary angioedema, thereby completing the present disclosure.

[0006] In some embodiments, the present disclosure provides an siRNA capable of inhibiting PKK gene expression, the siRNA comprising a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, the antisense strand comprises a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, and the nucleotide sequence I and the nucleotide sequence II are selected from the following sequences:

[0007] The nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2 and differs by no more than 3 nucleotides:

[0008] 5'-CUGAAUUCCAAAAACCAAZ1-3' (SEQ ID NO: 1);

[0009] 5'-Z2UUGGUUUUUGGAAUUCAG-3'(SEQ ID NO:2),

[0010] Among them, Z1 is U, Z2 is A,

[0011] Furthermore, the nucleotide sequence I contains a nucleotide Z3 corresponding to position Z1, and the nucleotide sequence II contains a nucleotide Z4 corresponding to position Z2, and Z4 is the first nucleotide at the 5' end of the antisense strand.

[0012] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the siRNA of the present disclosure and a pharmaceutically acceptable carrier.

[0013] In some embodiments, the present disclosure provides an siRNA conjugate, which contains the siRNA provided by the present disclosure and a conjugation group conjugated to the siRNA.

[0014] In some embodiments, the present disclosure provides use of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and / or preventing inflammatory diseases, particularly hereditary angioedema.

[0015] In some embodiments, the present disclosure provides a method for treating and / or preventing inflammatory diseases or embolic diseases or physiological conditions, particularly hereditary angioedema, comprising administering an effective amount of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure to a subject in need thereof.

[0016] In some embodiments, the present disclosure provides a method for inhibiting PKK gene expression in a cell, comprising contacting an effective amount of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure with the cell.

[0017] In some embodiments, the present disclosure provides a kit comprising the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure.

[0018] Incorporated by reference

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0020] Beneficial effects

[0021] The siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure have good stability, high PKKmRNA inhibitory activity, low off-target effects, and / or can treat, prevent or alleviate pathological conditions or disease symptoms caused by plasma kallikrein gene expression.

[0022] For example, at a siRNA concentration of 50 nM, the siRNA disclosed herein can achieve a 72.3% inhibition rate against PKK mRNA in primary liver cells of C57BL / 6J mice. In another example, the siRNA conjugates provided herein exhibited high inhibitory activity against PKK mRNA in mice, demonstrating a certain inhibitory effect against the PKK target sequence at various dosages, with an inhibition rate of up to 81.31%. In another example, the siRNA conjugates provided herein achieved an inhibition rate of up to 81.43% against PKK mRNA in mice modeling paw swelling.

[0023] For another example, the siRNA, pharmaceutical composition, or siRNA conjugate provided herein does not exhibit significant off-target effects. An off-target effect can be, for example, inhibition of normal gene expression of a non-target gene. It is generally considered that an off-target effect is not significant if the binding / inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% compared to the on-target gene effect.

[0024] For another example, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure shows a significant pharmacodynamic effect of inhibiting swelling. The siRNA conjugate provided by the present disclosure inhibits the expression of PKK mRNA and exhibits a dose-dependent inhibitory effect on carrageenan-induced mouse paw swelling, and this effect of inhibiting paw swelling is comparable to or even better than the inhibitory effect of the positive control drug. The siRNA conjugate provided by the present disclosure shows a high inhibitory activity on PKK mRNA in carrageenan-induced paw swelling model mice. At different dosages, the inhibition rate of PKK mRNA can reach more than 60%, among which at a dose of 3 mg / kg, the inhibition rate of PKK mRNA reaches 77.47%; at a dose of 9 mg / kg, the inhibition rate of PKK mRNA can reach 82.43%, showing a significant dose-dependent inhibition. However, the positive drugs indomethacin or icatibant did not significantly inhibit the expression of PKK mRNA. In terms of efficacy, the maximum swelling increase in mice with foot swelling model injected with normal saline reached 70.6%, while the maximum swelling increase in mice injected with the siRNA conjugate disclosed herein dropped significantly to below 39%. The time to reach the maximum swelling level was shorter, only 2 hours, and the swelling level subsequently subsided, indicating that the animals suffered less pain.

[0025] The above results indicate that the siRNA, pharmaceutical composition, and siRNA conjugate provided by the present disclosure can inhibit the expression of the PKK gene, effectively treat and / or prevent inflammatory diseases or embolic diseases or physiological conditions, especially hereditary angioedema and its related symptoms, and have good application prospects.

[0026] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a scatter plot of the relative expression levels of PKK mRNA in vivo in mice after administration of different doses of siRNA conjugates. DETAILED DESCRIPTION

[0028] The following describes the specific embodiments of the present disclosure in detail. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.

[0029] In the present disclosure, PKK mRNA refers to an mRNA having a sequence shown in Genbank Accession No. NM_008455.4, NM_001318394.1 or NM_001318396.1. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to a gene that transcribes the above-mentioned PKK mRNA, and the term "target mRNA" refers to the above-mentioned PKK mRNA.

[0030] definition

[0031] In the above and below texts, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorine-modified nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are connected by phosphorothioate groups; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide, the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate-modified nucleotide, the letter combination Ps indicates that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate-modified nucleotide, and capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0032] In the above and below, the term "fluorinated nucleotide" refers to a nucleotide in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by fluorine, and a "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2'-position of the ribose group of a nucleotide is replaced by a non-fluorinated group. A "nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. Examples include isonucleotides, bridged nucleic acids (BNAs), and acyclic nucleotides. The term "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0033] In the context of this article, the expressions "complementary" and "reverse complementary" are used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair consists of a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Accordingly, "mismatch" means in the art that the bases at corresponding positions in a double-stranded nucleic acid are not paired in a complementary manner.

[0034] In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there is no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there is no base mismatch between the two nucleotide sequences.

[0035] As used above and below, a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the base type of the nucleotide at the same position in the former compared to the latter. For example, if a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G, or T, the two nucleotide sequences are considered to have a nucleotide difference at that position. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, a nucleotide difference at that position can also be considered.

[0036] In the above and below, particularly when describing the preparation method of siRNA of the present disclosure, compositions containing siRNA or siRNA conjugates, unless otherwise specified, described nucleoside monomers (nucleoside monomers) refer to, according to the kind and order of nucleotides in the siRNA or siRNA conjugates to be prepared, the modification or unmodified nucleoside phosphoramidite monomers (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in phosphoramidite solid phase synthesis. Phosphoramidite solid phase synthesis is the method used in RNA synthesis well known to those skilled in the art. The nucleoside monomers used in the present disclosure all can be commercially available.

[0037] In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each of which has a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties with specific functions to siRNA. siRNA conjugates should be understood as the general term for siRNA conjugates, the general term for siRNA conjugates shown in formula (305) and formula (307), or the siRNA conjugates shown in formula (305), formula (307), and formula (308), depending on the context. In the context of the present disclosure, "conjugated molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction to ultimately form the siRNA conjugate of the present disclosure.

[0038] As used herein, a hyphen ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example: -C1-C 10 Alkyl-NH2 through C1-C 10 Alkyl group connected.

[0039] As used herein, "optional" or "optionally" means that the event or situation described thereafter may or may not occur, and the description includes both cases where the event or situation occurs and cases where it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. In the above or below, substituted groups, such as substituted alkyl, substituted alkoxy, substituted amino, substituted aliphatic group, substituted heteroaliphatic group, substituted acyl, substituted aryl or substituted heteroaryl. Wherein, unless otherwise specified, a "substituted" group refers to a group in which the hydrogen atoms in the group are replaced by one or more substituents. For example, "substituted alkoxy" refers to a group in which one or more hydrogen atoms in an alkoxy group are replaced by a substituent. It will be understood by those skilled in the art that various substituents may be contained in the compounds that can be used in the present disclosure, as long as the introduction of the substituent does not affect the function of the present disclosure and can achieve the purpose of the present disclosure, it can be used in the present disclosure. In some embodiments, the substituent is selected from the group consisting of the following groups: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 In some embodiments, the substituent is one of a C1-C3 alkyl group, a C6-C8 aryl group, a -OC1-C3 alkyl group, a -OC1-C3 alkylphenyl group, a halogen group, -OH, -NH2, a cyano group, or a nitro group. It will be understood by those skilled in the art that for any group comprising one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically infeasible, and / or inherently unstable.

[0040] As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms. When referring to an alkyl residue having a specific number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a residue that is the same as alkyl, but has two points of attachment.

[0041] As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by removing a molecule of hydrogen from the adjacent carbon atoms of the parent alkyl group. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In certain embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues identical to alkenyl but with two points of attachment.

[0042] As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond, obtained by removing two molecules of hydrogen from adjacent carbon atoms of a parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. In certain embodiments, alkynyl groups have 2 to 20 carbon atoms, and in other embodiments, have 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are identical to alkynyl, but with two points of attachment.

[0043] As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentoxy, etc. The alkoxy group typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through the oxygen bridge.

[0044] As used herein, "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2)π-electron system according to Hückel's theory. Aryl includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to residues identical to aryl but having two points of attachment.

[0045] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. The ring can be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.

[0046] As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo or iodo, and the term "halogen" includes fluorine, chlorine, bromine or iodine.

[0047] As used herein, "haloalkyl" refers to an alkyl group as defined above in which a specified number of carbon atoms is substituted with one or more, up to the maximum permitted number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0048] "Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical comprising 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise indicated in the specification, a heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system that may include fused or bridged ring systems. The heteroatoms in the heterocyclyl may optionally be oxidized. One or more nitrogen atoms, if present, may optionally be quaternized. The heterocyclyl may be partially saturated or fully saturated. The heterocyclyl may be attached to the remainder of the molecule via any of the ring atoms. Examples of such heterocyclic groups include, but are not limited to, dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0049] Various hydroxy protecting groups can be used in the present disclosure. In general, protecting groups make chemical functional groups insensitive to specific reaction conditions and can be added and removed on the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, each of which is incorporated herein by reference in its entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0050] The term "subject," as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, rabbits, and any type of poultry.

[0051] As used herein, "treat," "treat," "alleviate," or "amortize" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is achieved by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, even though the subject may still be afflicted with the underlying disorder.

[0052] As used herein, "prevent" and "prevention" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," siRNA, siRNA conjugate, or pharmaceutical composition can be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0053] siRNA

[0054] In one aspect, the present disclosure provides an siRNA capable of inhibiting PKK gene expression.

[0055] The siRNA disclosed herein contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be described in detail here.

[0056] The siRNA disclosed herein comprises a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2 and differs by no more than 3 nucleotides:

[0057] 5'-CUGAAUUCCAAAAACCAAZ1-3' (SEQ ID NO: 1);

[0058] 5'-Z2UUGGUUUUUGGAAUUCAG-3'(SEQ ID NO:2),

[0059] Among them, Z1 is U, Z2 is A,

[0060] Furthermore, the nucleotide sequence I contains a nucleotide Z3 corresponding to position Z1, and the nucleotide sequence II contains a nucleotide Z4 corresponding to position Z2, and Z4 is the first nucleotide at the 5' end of the antisense strand.

[0061] In the above and below, "corresponding position" means being at the same position in the nucleotide sequence, starting from the same end of the nucleotide sequence. For example, the first nucleotide from the 3' end of nucleotide sequence 1 is the nucleotide that corresponds to the first nucleotide from the 3' end of SEQ ID NO: 1.

[0062] In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.

[0063] In some embodiments, there is no more than one nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1, and / or there is no more than one nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2.

[0064] In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at position Z4, and Z4 is selected from U, C, or G. In some embodiments, the nucleotide difference is a difference at position Z4, and Z4 is selected from U, C, or G. In some embodiments, Z3 is a nucleotide complementary to Z4. siRNAs having the above-mentioned nucleotide differences have a higher ability to inhibit target mRNA, and these siRNAs containing nucleotide differences are also within the scope of protection of the present disclosure.

[0065] In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary; the substantially reverse complementary refers to that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary refers to that there is no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary refers to that there are no base mismatches between the two nucleotide sequences.

[0066] In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4:

[0067] 5'-CUGAAUUCCAAAAACCAAZ3-3' (SEQ ID NO: 3);

[0068] 5'-Z4UUGGUUUUUGGAAUUCAG-3'(SEQ ID NO:4),

[0069] wherein Z4 is the first nucleotide at the 5' end of the antisense strand, Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3; in some embodiments, Z3 is U and Z4 is A;

[0070] Furthermore, the sense strand and the antisense strand are of the same or different lengths, the sense strand is 19-23 nucleotides long, and the antisense strand is 19-26 nucleotides long. Thus, the ratio of the length of the sense and antisense strands of the siRNA provided by the present disclosure can be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is 19 / 21, 21 / 23, or 23 / 25.

[0071] In some embodiments, the sense strand further comprises nucleotide sequence III, and the antisense strand further comprises nucleotide sequence IV, wherein nucleotide sequence III and nucleotide sequence IV are each 1-4 nucleotides in length; nucleotide sequence III and nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; nucleotide sequence III is linked to the 5' end of nucleotide sequence I, and nucleotide sequence IV is linked to the 3' end of nucleotide sequence II. In some embodiments, nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which is a nucleotide sequence adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 1 in the target mRNA and has the same length as nucleotide sequence IV.

[0072] In some embodiments, in the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U; in this case, the length ratio of the sense chain and the antisense chain is 20 / 20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UA, and the base composition of the nucleotide sequence IV is UA; in this case, the length ratio of the sense chain and the antisense chain is 21 / 21; or, the length of the nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AUA, and the base composition of the nucleotide sequence IV is UAU; in this case, the length ratio of the sense chain and the antisense chain is 22 / 22; or, the length of the nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UAUA, and the base composition of the nucleotide sequence IV is UAUA; in this case, the length ratio of the sense chain and the antisense chain is 23 / 23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UA, and the base composition of the nucleotide sequence IV is UA; in this case, the length ratio of the sense chain and the antisense chain is 21 / 21.

[0073] In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the bases of nucleotide sequence III, the bases of nucleotide sequence IV are also determined.

[0074] In some embodiments, the antisense strand further comprises a nucleotide sequence V having a length of 1 to 3 nucleotides, which is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided herein can be 19 / 20, 19 / 21, 19 / 22, 20 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23, 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the length of the nucleotide sequence V is 2 nucleotides, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided herein can be 19 / 21, 21 / 23 or 23 / 25.

[0075] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate synthesis and save synthesis costs, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); alternatively, to improve the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding position of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA disclosed herein is 19 / 21 or 21 / 23. In this case, the siRNA disclosed herein has better target mRNA silencing activity.

[0076] The nucleotide at the corresponding position of the target mRNA refers to the nucleotide or nucleotide sequence adjacent to the 5' end of a nucleotide sequence of the target mRNA. The nucleotide sequence of the target mRNA segment is substantially reverse complementary or completely reverse complementary to Nucleotide Sequence II, or substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of Nucleotide Sequence II and Nucleotide Sequence IV.

[0077] In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 6:

[0078] 5'-CUGAAUUCCAAAAACCAAZ3-3' (SEQ ID NO:5);

[0079] 5'-Z4UUGGUUUUUGGAAUUCAGUA-3' (SEQ ID NO: 6);

[0080] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 8:

[0081] 5'-UACUGAAUUCCAAAAACCAAZ3-3' (SEQ ID NO:7);

[0082] 5'-Z4UUGGUUUUUGGAAUUCAGUAUA-3' (SEQ ID NO:8);

[0083] Wherein, Z4 is the first nucleotide at the 5' end of the antisense strand, Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3.

[0084] In some embodiments, the siRNAs disclosed herein are siPKKa1 and siPKKa2 listed in Table 1.

[0085] As previously described, the nucleotides in the siRNAs of the present disclosure are each independently modified or unmodified. In some embodiments, each nucleotide in the siRNAs of the present disclosure is an unmodified nucleotide; in some embodiments, some or all of the nucleotides in the siRNAs of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups do not significantly weaken or lose the function of the siRNAs of the present disclosure to inhibit PKK gene expression.

[0086] In some embodiments, the siRNA of the present disclosure contains at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide having a modified base. The modified nucleotide does not significantly weaken or lose the function of the siRNA to inhibit gene expression. For example, the modified nucleotide disclosed in JK Watts, GF Deleavey, and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discov. Today, 2008, 13 (19-20): 842-55 can be selected.

[0087] In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided herein is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group; in other words, at least a portion of the phosphate groups and / or ribose groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group having a modified group and / or a ribose group having a modified group.

[0088] In some embodiments, all nucleotides in the sense strand and / or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided herein is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0089] The inventors of the present disclosure surprisingly discovered that the siRNA disclosed herein achieved a high balance between stability in plasma and gene silencing efficiency in animal experiments.

[0090] In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, at least the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides.

[0091] In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, the number of fluorinated modified nucleotides in the nucleotide sequence I is no more than 5, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides; the number of fluorinated modified nucleotides in the nucleotide sequence II is no more than 7, and at least the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides.

[0092] In some embodiments, in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 or positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides.

[0093] In the context of the present disclosure, a "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (7). A "non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.

[0094] The nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorinated groups are well known to those skilled in the art. These nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.

[0095] In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is as shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (11):

[0096]

[0097] Nucleotide analogs are groups that can replace nucleotides in nucleic acids but have a structure different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0098] BNA refers to a constrained or inaccessible nucleotide. BNA can contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endo sugar condensed bridge structure. The bridge is usually incorporated into the 2'- and 4'-positions of the ribose to provide a 2',4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):

[0099]

[0100] Acyclic nucleotides are a type of nucleotide formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide can be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is represented by formula (15) and GNA is represented by formula (16):

[0101]

[0102] In the above formulae (15) and (16), R is selected from H, OH or alkoxy (O-alkyl).

[0103] An isonucleotide is a compound formed by a change in the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide can be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18):

[0104]

[0105] In the compounds of formula (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; and R is selected from H, OH, F or the non-fluorinated groups described above.

[0106] In some embodiments, the nucleotide analog is selected from one of an isonucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.

[0107] In the above and below, “fluorinated nucleotides”, “2’-fluorinated nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by fluorine” and “nucleotides having a 2’-fluorinated ribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (7) formed by replacing the 2’-hydroxyl group of the nucleotide with fluorine; “methoxy-modified nucleotides”, “2’-methoxy-modified nucleotides”, “nucleotides in which the 2’-hydroxyl group of the ribose group is substituted by a methoxy group” and “nucleotides having a 2’-methoxyribose group” have the same meaning, and all refer to compounds having a structure as shown in formula (8) formed by replacing the 2’-hydroxyl group of the ribose group of the nucleotide with a methoxy group.

[0108] In some embodiments, the siRNA disclosed herein is an siRNA having the following modifications: in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 or positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.

[0109] In some embodiments, the siRNA disclosed herein is an siRNA having the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA antisense strand are methoxy-modified nucleotides;

[0110] Alternatively, in the direction from the 5' end to the 3' end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II in the antisense strand of the siRNA are fluorinated nucleotides, and the nucleotides at the remaining positions of the siRNA antisense strand are methoxy-modified nucleotides;

[0111] Alternatively, in the direction from the 5' end to the 3' end, the 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides.

[0112] In some embodiments, the siRNA provided by the present disclosure is any one of siPKKa1-M1, siPKKa1-M2, siPKKa1-M3, siPKKa2-M1, siPKKa2-M2, and siPKKa2-M3 listed in Table 1.

[0113] siRNA with these modifications is not only low-cost but also makes it less susceptible to cleavage by ribonucleases in the blood, thereby increasing nucleic acid stability and making it more resistant to nuclease hydrolysis. Furthermore, these modified siRNAs have a high inhibitory activity against target mRNA.

[0114] In some embodiments, at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense strand and the antisense strand of the siRNA provided herein is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a phosphorothioate group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a phosphorothioate group having a structure as shown in formula (1):

[0115]

[0116] This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0117] In some embodiments, in the siRNA provided herein, the phosphorothioate linkage is present at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:

[0118] between the first and second nucleotides at the 5' end of the sense strand;

[0119] between the second and third nucleotides at the 5' end of the sense strand;

[0120] between the first and second nucleotides at the 3' end of the sense strand;

[0121] between the second and third nucleotides at the 3' end of the sense strand;

[0122] between the first and second nucleotides at the 5' end of the antisense strand;

[0123] between the second and third nucleotides at the 5' end of the antisense strand;

[0124] between the first and second nucleotides at the 3' end of the antisense strand; and

[0125] between the second and third nucleotides at the 3' end of the antisense strand.

[0126] In some embodiments, the siRNA provided by the present disclosure is any one of siPKKa1-M1S, siPKKa1-M2S, siPKKa1-M3S, siPKKa2-M1S, siPKKa2-M2S, and siPKKa2-M3S listed in Table 1.

[0127] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

[0128] Commonly used 5'-phosphate nucleotides or 5'-phosphate analogue-modified nucleotides are well known to those skilled in the art. For example, a 5'-phosphate nucleotide may have the following structure:

[0129]

[0130] For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analogue-modified nucleotides:

[0131]

[0132] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0133] In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a thiophosphate modified nucleotide as shown in formula (5).

[0134] In some embodiments, the siRNA provided by the present disclosure is any one of siPKKa1-M1P1, siPKKa1-M2P1, siPKKa1-M3P1, siPKKa2-M1P1, siPKKa2-M2P1, siPKKa2-M3P1, siPKKa1-M1SP1, siPKKa1-M2SP1, siPKKa1-M3SP1, siPKKa2-M1SP1, siPKKa2-M2SP1, and siPKKa2-M3SP1 listed in Table 1.

[0135] The inventors of the present disclosure unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also has higher target mRNA inhibitory activity.

[0136] The siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. Modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.

[0137] Pharmaceutical composition

[0138] In another aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA described above as an active ingredient and a pharmaceutically acceptable carrier.

[0139] The pharmaceutically acceptable carrier may be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine, PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate), ... phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethylmethacrylate), PDMAEMA) and one or more of their derivatives.

[0140] In some embodiments, there are no special requirements for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:(1-500). In some embodiments, the above weight ratio is 1:(1-50).

[0141] In some embodiments, the pharmaceutical composition may further include other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

[0142] The pH buffer may be a tris hydrochloride buffer with a pH value of 7.5-8.5 and / or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.

[0143] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. The content of the protective agent may be 0.01-30% by weight based on the total weight of the pharmaceutical composition.

[0144] The osmotic pressure regulator can be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmoles / kilogram (mOsm / kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.

[0145] In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to form a liquid preparation. The liquid preparation can be, but is not limited to, administered subcutaneously, intramuscularly, or intravenously, and can also be, but is not limited to, administered via spray to the lungs, or administered via spray through the lungs to other organs and tissues (such as the liver). In some embodiments, the pharmaceutical composition is administered intravenously.

[0146] In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a helper lipid and / or a pegylated lipid. Wherein, the organic amine, the helper lipid and the pegylated lipid may be selected from one or more of the amine-containing transfection compound or its pharmaceutically acceptable salt or derivative, the helper lipid and the pegylated lipid described in Chinese patent application CN103380113A (incorporated herein by reference in its entirety).

[0147] In some embodiments, the organic amine may be a compound described in CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:

[0148]

[0149] in:

[0150] Each X 101 or X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C1-C 20 hydrocarbon chain;

[0151] Each Y 101 or Z 101 Each independently is C=O, C=S, S=O, CH-OH or SO2;

[0152] Each R 101 、R 102 、R 103 、R 104 、R 105 、R 106 or R 107 are each independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;

[0153] x is an integer from 1 to 10;

[0154] n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; wherein, if m=p=0, then R 102 It is hydrogen;

[0155] And, if at least one of n or m is 2, then R 103 and the nitrogen in formula (201) to form a structure as shown in formula (202) or formula (203):

[0156]

[0157] wherein g, e, and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).

[0158] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 In some embodiments, R in formula (201) 101 and R 102 Each of is independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0159] In some embodiments, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following formulas (204) to (213):

[0160]

[0161] Wherein, in formula (204) to formula (213), g, e and f are each independently an integer of 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R 103 Possible points of attachment to the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve attachment to the nitrogen atom in formula (201).

[0162] Among them, the compound represented by formula (201) can be prepared according to the description in CN103380113A.

[0163] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215):

[0164]

[0165]

[0166] The helper lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative;

[0167] The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0168] In some embodiments, in the pharmaceutical composition, the molar ratio of the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).

[0169] In some embodiments, the pharmaceutical composition particles formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.

[0170] In some embodiments, the weight ratio of siRNA to total lipids (e.g., organic amines, helper lipids and / or PEGylated lipids) (weight / weight ratio) in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of siRNA of the present disclosure to total lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.

[0171] In some embodiments, the components of the pharmaceutical composition can be sold independently and can be in the form of a liquid preparation when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, simply by replacing the existing siRNA with the siRNA provided by the present disclosure; in some embodiments, it can be prepared according to the following method:

[0172] The organic amine, the helper lipid, and the PEGylated lipid are suspended in alcohol in the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at around room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, for example, ethanol.

[0173] The siRNA provided herein is dissolved in a buffered saline solution to obtain an siRNA aqueous solution. The concentration of the buffered saline solution is 0.05-0.5 M, for example, 0.1-0.2 M. The pH of the buffered saline solution is adjusted to 4.0-5.5, for example, 5.0-5.2. The amount of buffered saline solution used is such that the concentration of the siRNA does not exceed 0.6 mg / mL, for example, 0.2-0.4 mg / mL. The buffer salt is selected from one or more soluble acetates and soluble citrates, for example, sodium acetate and / or potassium acetate.

[0174] The lipid solution and the siRNA aqueous solution are mixed, and the resulting mixture is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome formulation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5), for example, 1:4.

[0175] The incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilized to obtain the pharmaceutical composition provided by the present disclosure, whose physicochemical parameters are pH 6.5-8, encapsulation efficiency not less than 80%, particle size 40-200 nm, polydispersity index not higher than 0.30, and osmotic pressure 250-400 mOsm / kg; for example, the physicochemical parameters can be pH 7.2-7.6, encapsulation efficiency not less than 90%, particle size 60-100 nm, polydispersity index not higher than 0.20, and osmotic pressure 300-400 mOsm / kg.

[0176] Concentration or dilution can be performed before, after, or simultaneously with impurity removal. Impurity removal can be performed using various existing methods, such as ultrafiltration at 100K Da using a tangential flow system or a hollow fiber column, with the ultrafiltration exchange solution being phosphate buffered saline (PBS) at pH 7.4. Sterilization can be performed using various existing methods, such as filtration sterilization using a 0.22 μm filter.

[0177] siRNA conjugates

[0178] In yet another aspect, the present disclosure provides an siRNA conjugate, comprising the above-mentioned siRNA and a conjugation group conjugated to the siRNA.

[0179] In general, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, or at the 5' end of the antisense strand, or in the internal sequence of the siRNA. In some embodiments, the conjugation site of the siRNA and conjugated group is at the 3' end of the siRNA sense strand.

[0180] In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.

[0181] In some embodiments, the siRNA and conjugated group can be linked via acid-labile or reducible chemical bonds. These bonds can degrade in the acidic environment of endosomes, freeing the siRNA. For non-degradable conjugations, the conjugated group can be attached to the sense strand of the siRNA to minimize the effect of conjugation on siRNA activity.

[0182] In some embodiments, the pharmaceutically acceptable targeting group can be a ligand commonly used in the field of siRNA administration, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0183] In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatocytes, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.

[0184] 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 hepatocyte 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 hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand capable of binding to an asialoglycoprotein receptor (ASGPR) on the liver surface. The types of these ligands are well known to those skilled in the art, and their function is generally to bind to specific receptors on the surface of target cells, mediating the delivery of siRNA connected to the ligand to the target cells.

[0185] In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialo serum mucin (ASOR) or asialo fetuin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0186] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives, and sialic acid.

[0187] In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, Sugar, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptaside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other selections of the ligand can be found in, for example, the description of CN105378082A, the entire disclosure of which is incorporated herein by reference.

[0188] 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. It should be understood that the monovalent, divalent, trivalent, and tetravalent herein refer to the molar ratio of the siRNA molecule to the galactose or N-acetylgalactosamine molecule in the siRNA conjugate after the siRNA molecule forms the siRNA conjugate with the conjugated group containing the galactose or N-acetylgalactosamine molecule as the targeting group. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described herein is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0189] The targeting group can be connected to the siRNA molecule via a suitable linker. Those skilled in the art can select a suitable linker based on the specific type of the targeting group. These linkers, the type of targeting group, and the mode of connection to the siRNA can be found in the disclosure of WO2015006740A2, which is incorporated herein by reference in its entirety.

[0190] In some embodiments, the linker in the siRNA conjugates of the present disclosure has a structure as shown in formula (301):

[0191]

[0192] Wherein, k is an integer from 1 to 3;

[0193] L A Having a structure including an amide bond as shown in formula (302), L B It has a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L C is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trishydroxymethylaminomethane;

[0194]

[0195] Among them, n 302 ,q 302 and p 302 Each independently represents an integer from 2 to 6, optionally, n 302 ,q 302 and p 302 Each independently is 2 or 3; n 303 An integer between 4 and 16. Optionally, n 303 is an integer between 8 and 12, Indicates the site of covalent attachment of a group.

[0196] In the joint, each L A Each of the targeting groups is connected via an ether bond and connected via L C The oxygen atom of the hydroxyl group in the part is connected with L C Partially form ether bonds and connect; L B Through the carbonyl group in formula (303) and L C The nitrogen atom of the amino group in the moiety forms an amide bond to connect, and is connected to the siRNA through the oxygen atom in formula (303) to form a phosphate bond or a phosphorothioate bond.

[0197] In some embodiments, the siRNA conjugate provided by the present disclosure has a structure as shown in formula (305):

[0198]

[0199] Wherein, Nu represents the siRNA provided by the present disclosure.

[0200] In some embodiments, the linker in the siRNA conjugates of the present disclosure has the structure shown in formula (306):

[0201]

[0202] Among them, n 306 is an integer from 0 to 3, each p 306 are independently an integer from 1 to 6, Indicates the site of covalent attachment of a group; the linking group is connected to the targeting group through an ether bond formed by the oxygen atom marked by *; the linking group is connected to the siRNA by forming a phosphate bond or a phosphorothioate bond by at least one of the oxygen atoms marked by #, and the remaining oxygen atoms marked by # are connected to hydrogen atoms to form hydroxyl groups, or are connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups;

[0203] In some embodiments, the siRNA conjugates of the present disclosure have a structure as shown in formula (307):

[0204]

[0205] Wherein, Nu represents the siRNA provided by the present disclosure.

[0206] In some embodiments, the siRNA conjugate has a structure as shown in formula (308):

[0207]

[0208] in:

[0209] n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

[0210] m1, m2 or m3 are independently an integer selected from 2-10;

[0211] R 10 、R 11 、R 12 、R 13 、R 14 or R 15 are each independently H, or selected from the group consisting of: C1-C 10 Alkyl, C1-C 10 Halogenated alkyl and C1-C 10 alkoxy;

[0212] R3 is a group represented by the structure of formula A59:

[0213]

[0214] wherein E1 is OH, SH or BH2, and Nu is the siRNA disclosed herein;

[0215] R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10alkylphenyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl);

[0216] Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene, C2-C 10 Alkynylidene, C6-C 10 Arylene, C3-C 18 Heterocyclylene and C5-C 10 wherein L1 may optionally have any one or more substituents selected from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 Heteroaryl, C1-C 10 Haloalkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C10 Alkyl)(C1-C 10 Alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON(C1-C 10 Alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -NH(C1-C 10 alkylphenyl), -CONH2, -NHC(O)(C1-C 10 Alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)C1-C 10 Alkyl, -SO2(C1-C 10 Alkyl), -SO2(phenyl), -SO2(C1-C 10 Halogenated alkyl), -SO2NH2, -SO2NH(C1-C 10 Alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 Alkyl), -NHSO2(phenyl) and -NHSO2(C1-C 10 haloalkyl).

[0217] In some embodiments, L1 can be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:

[0218]

[0219] wherein each j1 is independently an integer of 1-20; each j2 is independently an integer of 1-20; R' is C1-C 10 alkyl;

[0220] Ra is selected from the group consisting of formula A27-A45 or any combination thereof:

[0221]

[0222]

[0223] Rb is C1-C 10 alkyl;

[0224] Indicates the site of covalent attachment of a group.

[0225] It will be understood by those skilled in the art that although L is defined as a linear alkylene group for convenience, it may not be a linear group or may be named differently, such as an amine or alkenyl group resulting from the above-mentioned replacement and / or substitution. For the purposes of this disclosure, the length of L is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring (such as a heterocyclylene or heteroarylene group) resulting from the replacement of a carbon atom of the linear alkylene group is counted as one atom.

[0226] M1 represents a targeting group, and its definition and selectable range are the same as those of the above-mentioned targeting groups. In some embodiments, each M1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0227] When M1 is a ligand with affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3, and n3 can be an integer from 0 to 4, ensuring that the number of M1 targeting groups in the siRNA conjugate is at least 2. In some embodiments, n1 + n3 ≥ 2, which can result in the number of M1 targeting groups being at least 3, thereby facilitating the binding of the M1 targeting groups to the asialoglycoprotein receptor on the liver surface, thereby promoting the entry of the siRNA conjugate into cells via endocytosis. Experiments have shown that when the number of M1 targeting groups is greater than 3, the increased ease of binding of the M1 targeting groups to the asialoglycoprotein receptor on the liver surface is not significantly increased. Therefore, considering multiple factors such as ease of synthesis, structural / processing costs, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2-3.

[0228] In some embodiments, when m1, m2 or m3 are independently selected from integers of 2-10, the spatial positions between multiple M1 targeting groups can be made suitable for the binding of the M1 targeting groups to the asialoglycoprotein receptors on the liver surface. In order to make the siRNA conjugates provided by the present disclosure simpler, easier to synthesize and / or reduce costs, in some embodiments, m1, m2 or m3 are each independently an integer of 2-5. In some embodiments, m1=m2=m3.

[0229] Those skilled in the art will understand that when R 10 、R 11 、R 12 、R 13 、R 14 or R 15 Each independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl, and C1-C 10 In some embodiments, R 10 、R 11 、R 12 、R 13 、R 14 or R 15 Each is independently selected from H, methyl and ethyl. 10 、R 11 、R 12 、R 13 、R 14 and R 15 Both are H.

[0230] R3 is a group of the structure shown in formula A59, wherein E1 is OH, SH or BH2. Based on the easy availability of raw materials for preparation, in some embodiments, E1 is OH or SH.

[0231] The selection of R2 is to achieve the connection with the nitrogen atom on the nitrogen-containing skeleton and A59. In the context of this disclosure, "nitrogen-containing skeleton" refers to the one connected to R 10 、R 11 、R 12 、R 13 、R 14 and R 15 The carbon atoms and N atoms of the R2 group are connected to each other in a chain structure. Therefore, R2 can be any connecting group that can connect the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, when the siRNA conjugate shown in formula (308) is prepared by a solid-phase synthesis process, the R2 group needs to contain both a connecting site connected to the N atom on the nitrogen-containing skeleton and a connecting site connected to the P in R3. In some embodiments, the site in R2 that is connected to the N atom on the nitrogen-containing skeleton forms an amide bond with N, and the site that is connected to the P atom on R3 forms a phosphate bond with the P atom; in some embodiments, R2 can be B5, B6, B5' or B6':

[0232]

[0233] in, Indicates the site of covalent attachment of a group.

[0234] The value range of q2 can be an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0235] The role of L1 is to connect the M1 targeting group to the N on the nitrogen-containing skeleton, providing liver targeting function for the siRNA conjugate shown in formula (308). In some embodiments, L1 is selected from a connection combination of one or more of the formula A1-A26 groups. In some embodiments, L1 is selected from a connection combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13. In some embodiments, L1 is selected from a connection combination of at least two of A1, A4, A8, A10 and A11. In some embodiments, L1 is selected from a connection combination of at least two of A1, A8, and A10.

[0236] In some embodiments, L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments, L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms in length.

[0237] In some embodiments, each j1 is independently an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, each j2 is independently an integer of 2-10, and in some embodiments, each j2 is independently an integer of 3-5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, and Rb are each selected in formulas A1-A26 to achieve connection between the M1 targeting group and the N atom on the nitrogen-containing skeleton, and to make the spatial position between the M1 targeting groups more suitable for binding of the M1 targeting group to the liver surface asialoglycoprotein receptor.

[0238] In some embodiments, the siRNA conjugate has a structure represented by formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] In some embodiments, the P atom in formula A59 can be connected to any possible position in the siRNA sequence, for example, the P atom in formula A59 can be connected to any nucleotide of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is connected to any nucleotide of the siRNA sense strand. In some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand. The end refers to the first 4 nucleotides of the sense strand or antisense strand from one end. In some embodiments, the P atom in formula A59 is connected to the end of the siRNA sense strand or antisense strand; in some embodiments, the P atom in formula A59 is connected to the 3' end of the siRNA sense strand. In the case of being connected to the above-mentioned position of the siRNA sense strand, the siRNA conjugate shown in formula (308) can release a separate siRNA antisense strand when unwinding after entering the cell to block the process of PKK mRNA translation into protein and inhibit PKK gene expression.

[0247] In some embodiments, the P atom in formula A59 can be connected to any possible position on the nucleotide in the siRNA, for example, on the 5' position of nucleotide, the 2' position of nucleotide, the 3' position of nucleotide or the base of nucleotide. In some embodiments, the P atom in formula A59 can be connected to the 2' position, 3' position or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is connected on the oxygen atom formed after the dehydrogenation of the 3' hydroxyl of the 3' terminal nucleotide of the siRNA sense chain (now, the P atom in formula A59 can also be regarded as the P atom in the phosphate group contained in the siRNA), or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the siRNA sense chain, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl of the 5' terminal nucleotide of the siRNA sense chain.

[0248] The inventors of the present disclosure unexpectedly discovered that the siRNAs disclosed herein, while having significantly improved stability in plasma and low off-target effects, also exhibited high PKK mRNA silencing activity, and that siRNA conjugates containing these siRNAs exhibited even higher PKK mRNA silencing activity. Therefore, in some embodiments, the siRNA disclosed herein can be one of the siRNAs shown in Table 1.

[0249] Table 1 siRNA sequences disclosed herein

[0250]

[0251]

[0252]

[0253] Wherein, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide; the lowercase letter s indicates that the two nucleotides to the left of the letter are connected by a phosphorothioate group; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, P1 is VP, Ps, or P, which represents a specific modification, wherein the letter combination VP indicates that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide, the letter combination Ps indicates that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0254] In the siRNA or siRNA conjugate disclosed herein, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond. The non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond carries a negative charge and may exist in the form of a hydroxyl group or a sulfhydryl group. The hydrogen ions in the hydroxyl group or the sulfhydryl group may also be partially or completely replaced by cations. The cations may be any cations, such as metal cations, ammonium ions NH4 + , one of the organic ammonium cations. In order to improve solubility, in some embodiments, the cation is selected from one or more of alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations. The alkali metal ion can be K + and / or Na + The cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate described in the present disclosure can exist at least partially in the form of a salt. In some embodiments, the non-bridging oxygen atoms or sulfur atoms in the phosphodiester bond or phosphorothioate diester bond are at least partially bound to sodium ions, and the siRNA or siRNA conjugate described in the present disclosure exists in the form of a sodium salt or a partial sodium salt.

[0255] It is well known to those skilled in the art that modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared using known methods.

[0256] Preparation of siRNA conjugates disclosed herein

[0257] The above-mentioned siRNA conjugates can be synthesized by methods that have been described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various siRNA conjugates. The siRNA conjugates of the present disclosure are obtained by methods well known to those skilled in the art. For example, the preparation method of the structure shown in formula (305) is described in WO2014025805A1, and Rajeev et al. describe the preparation method of the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908. Chinese patent application CN110959011A also discloses in detail the method for preparing the siRNA conjugate shown in formula (308). The contents of the above-mentioned documents are incorporated herein by reference in their entirety.

[0258] The siRNA conjugates disclosed herein may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For details, please refer to the above description of the pharmaceutical composition disclosed herein.

[0259] Applications of the siRNA, pharmaceutical composition, and siRNA conjugate disclosed herein

[0260] In another aspect, the present disclosure provides use of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and / or preventing inflammatory diseases or embolic diseases or physiological conditions, in particular hereditary angioedema and its related symptoms.

[0261] In another aspect, the present disclosure provides a method for preventing and / or treating inflammatory diseases or embolic diseases or physiological conditions, in particular hereditary angioedema and its related symptoms, which comprises administering an effective amount of the siRNA and / or pharmaceutical composition and / or siRNA conjugate of the present disclosure to a subject in need thereof.

[0262] By administering the siRNA active ingredient disclosed herein to a subject in need thereof, the purpose of preventing and / or treating inflammatory diseases or embolic diseases or physiological conditions, particularly hereditary angioedema and its related symptoms, can be achieved through the mechanism of RNA interference. Therefore, the siRNA and / or pharmaceutical compositions and / or siRNA conjugates disclosed herein can be used to prevent and / or treat inflammatory diseases or embolic diseases or physiological conditions, particularly hereditary angioedema and its related symptoms, or to prepare a medicament for preventing and / or treating inflammatory diseases or embolic diseases or physiological conditions, particularly hereditary angioedema and its related symptoms.

[0263] In some embodiments, the inflammatory disease can be a chronic inflammatory disease or an acute inflammatory disease. In some embodiments, the inflammatory disease includes but is not limited to hereditary angioedema (HAE), edema, angioedema, swelling, angioedema of the eyelids, ocular edema, macular edema or cerebral edema. In some embodiments, the thromboembolic disease includes but is not limited to thrombosis, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, stroke or infarction.

[0264] The diseases may share one or more risk factors, causes, or consequences.

[0265] Certain risk factors and causes for developing inflammatory diseases include genetic predisposition to inflammatory diseases and environmental factors. In some embodiments, the subject has a mutated complement 1 esterase inhibitor (C1-INH) gene or a mutated factor 12 (FXII) gene. In some embodiments, the subject carries or has a tensin-converting enzyme inhibitor (ACE inhibitor) or tensin II receptor blocker (ARB). In some embodiments, the subject has had an allergic reaction resulting in angioedema. In some embodiments, the subject has type I HAE. In some embodiments, the subject has type II HAE. In some embodiments, the subject has type III HAE. Certain outcomes associated with the development of inflammatory diseases include edema / swelling of various body parts, including extremities (i.e., hands, feet, arms, legs), intestines (abdominal cavity), face, genitals, larynx (i.e., larynx); vascular permeability; vascular leak; systemic inflammation; abdominal pain; bloating; vomiting; diarrhea; itchy skin; respiratory (asthmatic) reactions; rhinitis; anaphylaxis; bronchoconstriction; hypotension; coma, and death.

[0266] Certain risk factors and causes for developing thromboembolic disease include a genetic predisposition to thromboembolism, surgery (particularly orthopedic surgery), malignancy, pregnancy, advanced age, use of oral contraceptives, atrial fibrillation, prior thromboembolic conditions, chronic inflammatory diseases, and inherited or acquired coagulation disorders that prevent thromboembolic agents. Certain consequences associated with the development of thromboembolic conditions include decreased blood flow through the affected blood vessels, tissue death, and death.

[0267] As used herein, the term "administration / administration" refers to placing the siRNA, pharmaceutical composition and / or siRNA conjugate of the present disclosure into the body of a subject by a method or route that at least partially localizes the siRNA, pharmaceutical composition and / or siRNA conjugate of the present disclosure to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in more siRNA conjugates being delivered to a specific site compared to the systemic circulation of the subject; while systemic administration results in the delivery of the siRNA, pharmaceutical composition and / or siRNA conjugate of the present disclosure to the general systemic circulation of the subject. Given that the present disclosure is intended to provide a means for preventing and / or treating inflammatory or embolic diseases or physiological conditions, particularly hereditary angioedema and its associated symptoms, in some embodiments, a mode of administration that is capable of delivering the drug to the liver is employed.

[0268] The drug may be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal administration and sublingual administration). The frequency of administration may be once or more daily, weekly, biweekly, three-weekly, monthly, bimonthly, quarterly, semi-annually, or annually.

[0269] The dosage of the siRNA, pharmaceutical composition or siRNA conjugate described in the present disclosure can be a conventional dosage in the art, which can be determined based on various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determining LD 50 (lethal dose that kills 50% of the group) and ED 50 (For quantitative responses, this refers to the dose that induces a 50% maximal response; for qualitative responses, it refers to the dose that induces a positive reaction in 50% of the subjects.) A range of human dosage can be derived based on data from cell culture assays and animal studies.

[0270] When administering the siRNA, pharmaceutical composition, and / or siRNA conjugate described in the present disclosure, for example, for male or female C57BL / 6J or C3H / HeNCrlVr mice aged 6-12 weeks and weighing 18-25 g, based on the amount of siRNA: (i) for siRNA conjugates, the amount of siRNA can be 0.001-100 mg / kg body weight, in some embodiments, 0.01-50 mg / kg body weight, in some embodiments, 0.05-20 mg / kg body weight, in other embodiments, 0.1-15 mg / kg body weight, and in other embodiments, 0.1-10 mg / kg body weight; (ii) for pharmaceutical compositions formed by siRNA and a pharmaceutically acceptable carrier, the amount of siRNA can be 0.001-50 mg / kg body weight, in some embodiments, 0.01-10 mg / kg body weight, in some embodiments, 0.05-5 mg / kg body weight, and in some embodiments, 0.1-3 mg / kg body weight.

[0271] In addition, by introducing the siRNA and / or pharmaceutical composition and / or siRNA conjugate disclosed herein into cells, the purpose of inhibiting PKK gene expression in cells can also be achieved through the mechanism of RNA interference.

[0272] Adopt the method that the disclosure provides to suppress PKK gene expression in cell, the siRNA dosage in the siRNA that provides, pharmaceutical composition and / or siRNA conjugate is generally such amount: it is enough to reduce the expression of target mRNA, and causes the extracellular concentration of 1pM to 1 μ M or 0.01nM to 100nM or 0.05nM to 50nM or 0.05nM to about 5nM at target cell surface.The amount required for reaching this local concentration will change with various factors, and the factor comprises delivery method, delivery site, the number of the cell layer between delivery site and target cell or tissue, delivery route (local or whole body) etc.The concentration at delivery site can be significantly higher than the concentration at the surface of target cell or tissue.

[0273] Reagent test kit

[0274] In yet another aspect, the present disclosure provides a kit comprising an effective amount of at least one of the siRNA, pharmaceutical composition, and siRNA conjugate of the present disclosure.

[0275] In some embodiments, the kits described herein may provide the siRNA in a container. In some embodiments, the kits described herein may include a container for providing a pharmaceutically acceptable excipient. In some embodiments, the kits may also include other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein may include at least one other therapeutic agent in a container other than the container providing the siRNA described herein. In some embodiments, the kits may include instructions for mixing the siRNA with a pharmaceutically acceptable carrier and / or excipient or other ingredients (if any).

[0276] In the kit of the present disclosure, the siRNA and pharmaceutically acceptable carrier and / or adjuvant and the siRNA, pharmaceutical composition and / or siRNA conjugate, and / or pharmaceutically acceptable adjuvant can be provided in any form, such as liquid form, dried form or lyophilized form. In some embodiments, the siRNA and pharmaceutically acceptable carrier and / or adjuvant and the pharmaceutical composition and / or siRNA conjugate and optional pharmaceutically acceptable adjuvant are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0277] The present disclosure will be further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0278] Example

[0279] Unless otherwise specified, all reagents and culture media used in the following examples are commercially available products, and nucleic acid electrophoresis, real-time PCR and other operations used are performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0280] C57BL / 6J mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and are referred to as C57 mice hereafter;

[0281] When the siRNA, siRNA conjugate or siRNA or siRNA conjugate synthesized by the present disclosure against PKK gene or negative control is transfected into cells, Lipofectamine TM 2000 (Invitrogen) was used as the transfection reagent, and the specific operation was referred to the instructions provided by the manufacturer.

[0282] Unless otherwise stated, the reagent ratios provided below are calculated by volume (v / v).

[0283] Preparation Example 1

[0284] Preparation of siRNA conjugate 1

[0285] This preparation example synthesized siRNA conjugate 1. This siRNA conjugate is formed by conjugating the L-9 conjugate molecule with the siRNA numbered siPKKa1M1S. The sequence of the siRNA conjugated in this siRNA conjugate is shown in Table 2.

[0286] According to the method for preparing conjugate 16 described in Preparation Example 14 of CN110959011A, siRNA conjugate 1 in Table 2 below was prepared, with the only difference being that the sense and antisense strands of the siRNA contained in siRNA conjugate 1 are as shown in Table 2; the sense and antisense strands of the siRNA were synthesized according to the nucleic acid sequence of the siRNA numbered L10-siPKKa1M1S in Table 2 below. After siRNA conjugate 1 was diluted to a concentration of 0.2 mg / mL (based on siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). Specifically,

[0287] The molecular weight of siRNA conjugate 1 was 7507.51 for the sense chain and 7506.73 for the measured value. The molecular weight of the antisense chain was 6987.64 for the measured value and 6987.56 for the measured value.

[0288] The measured values ​​are consistent with the theoretical values, indicating that the synthesized siRNA conjugate 1 is the designed double-stranded nucleic acid sequence. siRNA conjugate 1 has the structure shown in formula (403), and the siRNA contained in the siRNA conjugate has the siRNA sequence corresponding to siRNA conjugate 1 in Table 2.

[0289] Table 2 siRNA conjugates

[0290]

[0291] Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group.

[0292] Preparation Example 2-5

[0293] Synthesis of siRNA provided by the present disclosure

[0294] The sense strand or antisense strand of the siRNA sequence provided by the present disclosure listed in Table 3 was obtained by conventional solid-phase synthesis method. DEPC water was used to dissolve equimolar complementary sense strands and antisense strands in Table 3, and then annealed to obtain the siRNA provided by the present disclosure, which were respectively designated as siPKKa1M1S, siPKKa2M4, siPKKa and siPKKa3, and their sequences are shown in Table 3.

[0295] Comparative Preparation Example 1

[0296] Synthesis of reference siRNA

[0297] The sense and antisense strands corresponding to the siRNAs designated NC in Table 3 were synthesized using solid-phase synthesis. Equimolar amounts of the sense and antisense strands were dissolved in DEPC water and then annealed to yield a reference siRNA designated NC. NC siRNAs are double-stranded oligonucleotides that do not target any known human, mouse, or rat genes.

[0298] Table 3 siRNA sequences

[0299]

[0300]

[0301] Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group.

[0302] The molecular weight of the above siRNA was tested according to the method of Preparation Example 1.

[0303] The molecular weight of Preparation Example 2 was 6259.23 for the sense strand and 6259.01 for the antisense strand; 6987.64 for the antisense strand and 6987.56 for the antisense strand.

[0304] The molecular weight of Preparation Example 3 was 6764.30 for the sense chain and 6764.12 for the antisense chain; 7418.50 for the antisense chain and 7418.96 for the antisense chain.

[0305] The molecular weight of Preparation Example 4 was 5996.70 for the sense chain and 5996.33 for the antisense chain; 6041.59 for the antisense chain and 6041.22 for the antisense chain.

[0306] The molecular weight of Preparation Example 5 was 6655.12 for the sense chain theoretical value and 6655.02 for the measured value, and 7289.30 for the antisense chain theoretical value and 7289.11 for the measured value.

[0307] The measured values ​​were consistent with the theoretical values, confirming that the obtained siRNAs had sequences corresponding to the respective siRNAs shown in Table 3.

[0308] After the siRNA or conjugate disclosed above is prepared, it is lyophilized into a solid powder and stored for future use.

[0309] Experimental Example 1

[0310] Determination of the inhibition rate of PKK mRNA in mouse liver primary cells by siRNA provided by the present disclosure

[0311] Mouse primary liver cells were obtained from fresh liver tissue of C57BL / 6J mice, seeded on type I collagen-coated tissue culture dishes, and cultured in RPMI1640 medium containing 1× double antibody and 10% FBS at 37°C in an incubator containing 5% CO2 / 95% air for 30 min.

[0312] The culture medium was discarded and the density of mouse liver primary cells was adjusted to 2×10 5cells / mL to obtain a mouse liver primary cell suspension. Subsequently, the obtained mouse liver primary cell suspension was added to different culture wells of a 12-well plate, and the mouse liver primary cells were inoculated into the culture wells. The volume of the mouse liver primary cell suspension added was 1 mL / well, and the number of mouse liver primary cells was 2×10 5 cells / well.

[0313] The siRNAs obtained in Preparation Examples 2-5 and Comparative Preparation Example 1 were respectively prepared into 50 μM siRNA working solutions using DEPC water.

[0314] Prepare 1A solution. For each siRNA, prepare 1A1-1A3 solutions respectively. Each 1A1-1A3 solution contains 1.1 μl of the above siRNA working solution and 50 μl of Opti-MEM medium.

[0315] Prepare 1B solution, each 1B solution contains 1μl Lipofectamine TM 2000 and 50 μl Opti-MEM medium.

[0316] Separately mix a portion of 1B solution with the obtained 1A solution of each siRNA, and incubate at room temperature for 20 minutes to obtain a 1X transfection complex of each siRNA.

[0317] A portion of 1B solution was mixed with 50 μl of Opti-MEM medium and incubated at room temperature for 20 min to obtain transfection complex 1X'.

[0318] Add 1X transfection complex of each siRNA to the culture wells, mix evenly, and add 100 μl / well to obtain a transfection complex with a final concentration of approximately 50 nM for each siRNA. Transfect 3 culture wells with 1X transfection complex of each siRNA to obtain a transfection mixture containing siRNA, which is recorded as the test group.

[0319] In the other three culture wells, 1X' transfection complex was added at a volume of 100 μl / well to obtain a transfection mixture without siRNA, which was recorded as a blank control group.

[0320] After 4 hours of transfection with the siRNA-containing and non-siRNA-containing transfection mixtures in the culture wells, 1 ml of H-DMEM complete medium containing 20% ​​FBS was added to each well, and the 12-well plates were placed in a CO2 incubator and cultured for another 24 hours at 37°C.

[0321] Subsequently, total RNA from the cells in each well was extracted using RNAVzol (purchased from Vigoras Biotechnology (Beijing) Co., Ltd., catalog number N002) according to the method described in the instruction manual.

[0322] For each well of cells, 1 μg of total RNA was taken and reverse transcription kit Goldenstar was used. TM The reagents provided by RT6cDNASynthesis Kit (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd., product number TSK301M) were selected from Goldenstar TM Oligo(dT) 17 As primers, prepare 20 μl of reverse transcription reaction system according to the reverse transcription protocol in the kit instructions, and reverse transcribe the total RNA of the cells in each well. Reverse transcription conditions are as follows: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 50°C for 50 minutes, then at 85°C for 5 minutes, and finally at 4°C for 30 seconds. After the reaction is completed, add 80 μl of DEPC water to the reverse transcription reaction system to obtain a solution containing cDNA.

[0323] For each reverse transcription reaction system, 5 μl of the above solution containing cDNA was taken as template and used A 20 μl qPCR reaction system was prepared using the reagents provided in the SYBR qPCR SuperMix Plus kit (purchased from Jinan Protein Technology Co., Ltd., Cat. No. E096-01B). The PCR primer sequences for amplifying the target gene PKK and the internal reference gene GAPDH are shown in Table 4, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step protocol: a 95°C initial denaturation for 10 min, followed by a 95°C denaturation for 30 s, a 60°C annealing for 30 s, and a 72°C extension for 30 s. This denaturation, annealing, and extension cycle was repeated 35 times to obtain product W1 containing the amplified target gene PKK and the internal reference gene GAPDH. The product W1 was then incubated at 95°C for 15s, 60°C for 1min, and 95°C for 15s. The melting curves of the target gene PKK and the internal reference gene GAPDH in the product W1 were collected by real-time fluorescence quantitative PCR instrument to obtain the Ct values ​​of the target gene PKK and the internal reference gene GAPDH.

[0324] Table 4 Primer information

[0325]

[0326] The comparative Ct (ΔΔCt) method was used to calculate the relative quantification of the target gene PKK in each test group. The calculation method is as follows:

[0327] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0328] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0329] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0330] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0331] Wherein, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) of each of the three culture wells of the control group. Thus, each culture well of the test group and the control group corresponds to a ΔΔCt value.

[0332] The expression level of PKK mRNA in the test group was normalized with the control group as the benchmark, and the expression level of PKK mRNA in the blank control group was defined as 100%.

[0333] The relative expression level of PKK mRNA in the test group = 2 -ΔΔCt(测试组) ×100%

[0334] PKK mRNA inhibition rate in the test group = (1-relative expression level of PKK mRNA in the test group) × 100%

[0335] The inhibition rate of each siRNA on PKK mRNA is summarized in Table 5. For the same test group of siRNA, the PKK mRNA inhibition rate is the arithmetic mean of the PKK mRNA inhibition rates of the test group measured in three culture wells.

[0336] Table 5 Inhibition of PKK mRNA in primary mouse liver cells

[0337]

[0338] As can be seen from the results in Table 5, the siRNA provided by the present disclosure exhibited high PKK mRNA inhibitory activity in mouse primary liver cells when the siRNA had different lengths, mismatches between the antisense chain and the mRNA, or different modifications. At an siRNA concentration of 50 nM, the PKK mRNA inhibition rates of Preparation Examples 2-5 were all higher than 60.0%, among which the PKK mRNA inhibition rate of Preparation Example 2 reached 72.3%.

[0339] Experimental Example 2

[0340] Determination of in vivo activity of the siRNA conjugates provided by the present disclosure in C57BL / 6J mice

[0341] C57BL / 6J mice were randomly divided into groups of 5 (all male) and administered different doses of siRNA conjugate 1. All animals were dosed according to body weight and administered a single subcutaneous injection. siRNA conjugate 1 was administered at 0.1 mg / ml and 0.3 mg / ml of a 0.9 wt% sodium chloride aqueous solution in a volume of 10 ml / kg of mouse body weight, resulting in doses of 1 mg / kg and 3 mg / kg, respectively. Additionally, 10 male C57BL / 6J mice served as a control group and were administered saline in a 0.9 wt% sodium chloride aqueous solution in a volume of 10 ml / kg of mouse body weight. Eight days after administration, mice were sacrificed, and plasma was collected. 3.2 wt% (0.109 mol / L) sodium citrate dihydrate aqueous solution was added at an anticoagulant to plasma ratio of 1:9 (v / v) to prevent coagulation, and the plasma was separated by centrifugation.

[0342] About 100 mg of the left lobe of the liver was collected from each mouse and stored in RNA later (Sigma Aldrich). The liver tissue of each mouse was then homogenized using a tissue homogenizer and then extracted using Trizol (Thermo Fisher) according to the instructions. ImProm-II TM The extracted total RNA was reverse transcribed using a reverse transcription kit (Promega) according to the manufacturer's instructions. The reverse transcription conditions were as follows: incubate the reaction system at 50°C for 50 minutes, then at 85°C for 5 minutes, and finally at 4°C for 30 seconds. After the reaction, 80 μl of DEPC water was added to the reaction system to obtain a solution containing cDNA. PKK mRNA expression in liver tissue was then detected using a fluorescent quantitative PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.).

[0343] For each reverse transcription reaction system, 5 μl of the above solution containing cDNA was taken as template and used The reagents provided in the SYBR qPCR SuperMix Plus kit (purchased from Jinan Protein Technology Co., Ltd., Cat. No. E096-01B) were used to prepare a 20 μl qPCR reaction system. The PCR primer sequences for amplifying the target gene PKK and the internal reference gene GAPDH are shown in Table 6, with a final concentration of each primer of 0.25 μM. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument and amplified using a three-step protocol: a 10-min initial denaturation at 95°C, followed by a 30-s denaturation at 95°C, a 30-s annealing at 60°C, and a 30-s extension at 72°C. This denaturation, annealing, and extension cycle was repeated 40 times to obtain product W2 containing the amplified target gene PKK and the internal reference gene GAPDH. Product W2 was then incubated at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s, and the melting curves of the target gene PKK and the internal reference gene GAPDH in product W2 were measured by real-time fluorescence quantitative PCR to obtain the Ct values ​​of the target gene PKK and the internal reference gene GAPDH.

[0344] Table 6 Sequences of detection primers

[0345]

[0346] The comparative Ct (ΔΔCt) method was used to perform relative quantitative calculation of the expression level of the target gene PKK in each test group and the control group. The calculation method is as follows:

[0347] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0348] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0349] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0350] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0351] Here, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) values ​​of the 10 mice in the control group. Thus, each test mouse in the test group and the control group corresponds to a ΔΔCt value.

[0352] The expression level of PKK mRNA in the test group was normalized with the control group as the benchmark, and the expression level of PKK mRNA in the saline control group was defined as 100%.

[0353] The relative expression level of PKK mRNA in the test group = 2- ΔΔCt(测试组)×100%.

[0354] For the same test group siRNA, the average relative expression level of PKK mRNA in the test group at each concentration is the arithmetic mean of the relative expression levels of 5 mice at that concentration. Graphpad prism 6.0 statistical analysis software was used for data analysis. The PKK mRNA expression level of the control group was recorded as 100%, and accordingly, the PKK mRNA expression inhibition rate was recorded as 0%. The test results were normalized with the PKK mRNA expression level of the control group, and the results are shown in Table 7. In Table 7, the dosage was calculated based on siRNA. The control group was not given siRNA, and the corresponding dosage column was marked with "-". The PKK mRNA inhibition rate was the average PKK mRNA inhibition rate of a group of mice given the corresponding siRNA conjugate:

[0355] PKK mRNA inhibition rate = (1-PKK mRNA relative expression level) × 100%.

[0356] Table 7 Inhibition rate of PKK mRNA by the siRNA conjugates disclosed herein in mice

[0357]

[0358] The results of the relative expression levels of PKK mRNA in vivo after administration of different doses of siRNA conjugates are shown in Figure 1 middle.

[0359] From Table 7 and Figure 1 The results show that the siRNA conjugate 1 disclosed above showed a good PKK mRNA inhibitory effect in C57BL / 6J mice. Compared with the control group saline, the inhibition rate of PKK mRNA reached 81.31% when the siRNA conjugate 1 was administered at a dose of 3 mg / kg.

[0360] Experimental Example 3

[0361] Study on the efficacy of the siRNA conjugate provided by the present disclosure on the carrageenan-induced mouse paw swelling model

[0362] Carrageenan is a mucopolysaccharide derived from the cell walls of red algae and comes in various types. λ-carrageenan, among them, can be in a non-gelling state at room temperature. Subcutaneous injection can cause inflammatory reactions such as edema, increased capillary permeability, hyperalgesia, and erythema, and is used to induce a rodent paw swelling model. In this experimental example, mice were given different doses of siRNA conjugate 1 and the positive controls indomethacin and icatibant. Subsequently, λ-carrageenan was used to induce paw swelling in the mice. The extent to which the siRNA conjugates provided herein inhibited the increase in paw swelling was determined by the water displacement method, and the expression level of PKK mRNA in the mice was detected by fluorescent quantitative PCR.

[0363] In this experimental example, saline is a 0.9wt% sodium chloride aqueous solution purchased from Jiangxi Kelun Pharmaceutical Co., Ltd. (Batch No. C19070906). Before administration, siRNA conjugate 1 in Preparation Example 1 was prepared into aqueous solutions of 0.2mg / ml, 0.6mg / ml, and 1.8mg / ml, respectively, using saline. The positive control indomethacin was purchased from Sigma-Aldrich (Article No. I7378, Batch No. WXBC4210V) and was prepared into a 2mg / ml solution using 0.5wt% CMC-Na (sodium carboxymethyl cellulose, purchased from Sinopharm Chemical Reagent Effective Company, Article No. 30036358, Batch No. 20180412). The positive control icatibant acetate was purchased from MedChemExpress (Article No. HY-108896, Batch No. 81711) and was prepared into a 1.2mg / ml solution using saline. Indomethacin is a nonsteroidal anti-inflammatory drug that can relieve swelling and pain in joints and soft tissues; icatibant acetate is a potent and selective competitive antagonist of the bradykinin type 2 (B2) receptor, used to treat acute attacks of hereditary angioedema (HAE) in adults, adolescents and children ≥2 years of age.

[0364] Thirty male ICR mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were randomly divided into six groups of five mice each, designated G1-G6. The drug dose for all animals was calculated based on body weight, with a subcutaneous injection or oral gavage volume of 5 ml / kg of mouse body weight. The dosing schedule for each group of mice was as follows:

[0365] The mice in group G1 were subcutaneously injected with saline 7 days before the injection of λ-carrageenan solution (denoted as D-7), with the administration volume being 5 ml / kg of mouse body weight.

[0366] The mice in group G2 were orally administered with indomethacin at a dose of 10 mg / kg mouse body weight 1 hour before the injection of λ-carrageenan solution.

[0367] The mice in group G3 were subcutaneously injected with icatibant at a dose of 6 mg / kg mouse body weight 1 hour before the injection of λ-carrageenan solution.

[0368] Seven days before the injection of λ-carrageenan solution, mice in groups G4-G6 were subcutaneously injected with different doses of siRNA conjugate 1, with the dosages being 1 mg / kg, 3 mg / kg, and 9 mg / kg of mouse body weight, respectively.

[0369] Mice in the above groups that had been injected with different drugs were subcutaneously injected with a λ-carrageenan solution into the right hind footpad to induce foot swelling. λ-carrageenan was purchased from Sigma-Aldrich (Cat. No. 22049) and prepared in saline to a 1 wt% concentration. The injection volume was 40 μL per mouse. The day of λ-carrageenan injection was considered day 1 (D1).

[0370] One hour before injection, and two, four, and six hours after injection, the degree of swelling of the hind paws of each animal was measured by the water displacement method. The measurement method is as follows: 70 ml of distilled water was injected into a 100 ml beaker and zeroed on the electronic balance. Before the test, a measurement line was marked at the ankle joint of the mouse. Fix the mouse and place the mouse's hind paws into the beaker. When the horizontal plane overlaps with the measurement line at the ankle joint of the mouse, read the result on the balance display. The reading in grams (g) is the volume of water (ml) discharged after the mouse's paws are immersed in water, which indicates the degree of swelling of the mouse's paws. Each time the same mouse is measured, the position of the hind paws immersed in the distilled water in the beaker is the same, that is, the measurement line marked at the ankle joint overlaps with the horizontal plane. Re-zero after each measurement.

[0371] The degree of paw swelling, maximum swelling increase, and time to maximum swelling of each group of animals are shown in Table 8. In Table 8, maximum swelling increase of a group = (maximum swelling of the group - swelling of the group 1 hour before injection) / swelling of the group 1 hour before injection × 100%; time to maximum swelling of a group refers to the time to maximum swelling after injection of the λ-carrageenan solution, and maximum swelling refers to the maximum swelling of animals in a group.

[0372] After the last measurement of paw swelling, the animals were anesthetized by inhaling 2-5 v / v% isoflurane (purchased from Shenzhen Ruiwode Technology Co., Ltd., batch number: 20120301). The animals were then euthanized by inhaling excessive carbon dioxide and dislocating the cervical spine. For each mouse, 100 mg of the left lobe of the liver was removed, cut into 2×2 mm pieces, and preserved with RNA later (Sigma Aldrich). The expression of PKK mRNA in the liver tissue was detected using the same method as in Experimental Example 2. The inhibition rate of PKK mRNA in the carrageenan-induced paw swelling model mice is shown in Table 9.

[0373] During the entire experimental period, the animals showed no abnormal appearance or behavior, and there were no significant differences among the groups.

[0374] Table 8 Foot swelling degree of animals in each group (g)

[0375]

[0376] As can be seen from Table 8, 2 hours, 4 hours, and 6 hours after the injection of the λ-carrageenan solution, the mice in groups G1-G6 all developed varying degrees of foot swelling relative to their own pre-injection levels. The maximum swelling increase in group G1, which was injected with normal saline, reached 70.6%, while the maximum swelling increase in groups G2-G6, which were injected with different drugs, was significantly reduced to below 39%. On the other hand, the time it took for mice in groups G4-G6, which were injected with different doses of the siRNA conjugate 1 of the present disclosure, to reach the maximum swelling level was relatively short, all at 2 hours, and then the swelling level subsided. The degree of swelling inhibition was also significantly dose-related, indicating that the siRNA conjugate provided by the present disclosure has a significant and rapid effect in inhibiting mouse swelling.

[0377] Compared with the other two positive control drugs (Groups G2 and G3), the siRNA conjugates provided by the present disclosure have similar or better effects.

[0378] Table 9 Inhibition rate of PKK mRNA in each group of animals

[0379]

[0380]

[0381] As shown in Table 9, in the carrageenan-induced paw swelling model mice, the disclosed siRNA conjugate 1 demonstrated a significant inhibitory effect on PKK mRNA in mice. Compared to the G1saline group, the siRNA conjugate 1 achieved an inhibition rate of over 60% on PKK mRNA. At a dose of 3 mg / kg, the inhibition rate reached 77.47%; at a dose of 9 mg / kg, the inhibition rate reached 82.43%, demonstrating a clear dose-dependent inhibition. The positive control group, G2-G3, showed no significant inhibition on PKK mRNA expression. This indicates that the disclosed siRNA conjugate 1 significantly inhibited the increase in paw swelling by inhibiting PKK mRNA expression, demonstrating promising application prospects.

[0382] Some embodiments of the present disclosure are described in detail above. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.

[0383] It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0384] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.

Claims

1. An siRNA comprising a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified nucleotide, wherein: The sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary or completely reverse complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and differs by no more than one nucleotide, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 2: 5'-CUGAAUUCCAAAAACCAAZ1-3' (SEQ ID NO: 1); 5'-Z2UUGGUUUUUGGAAUUCAG-3'(SEQ ID NO:2), Among them, Z1 is U, Z2 is A, The nucleotide sequence I comprises a nucleotide Z3 corresponding to position Z1, the nucleotide sequence II comprises a nucleotide Z4 corresponding to position Z2, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 comprises a difference at position Z4, and Z4 is selected from U, C or G, Z4 is the first nucleotide at the 5' end of the antisense strand, and Z3 is a nucleotide complementary to Z4, The term "substantially reverse complementary" means that there is no more than one base mismatch between the two nucleotide sequences; and "perfectly reverse complementary" means that there is no mismatch between the two nucleotide sequences. Position correspondence refers to the same position in the nucleotide sequence, starting from the same end of the nucleotide sequence. Each nucleotide in the sense strand and the antisense strand is independently a fluorinated-modified nucleotide or a non-fluorinated-modified nucleotide, and the fluorinated-modified nucleotide is located in nucleotide sequence I and nucleotide sequence II, and, in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated-modified nucleotides; in the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated-modified nucleotides, and each non-fluorinated-modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.

2. The siRNA according to claim 1, wherein The nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 3, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 4: 5'-CUGAAUUCCAAAAACCAAZ3-3' (SEQ ID NO: 3); 5'-Z4UUGGUUUUUGGAAUUCAG-3'(SEQ ID NO:4), Wherein, Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3.

3. The siRNA according to claim 1 or 2, wherein The sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV. The lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 1-4 nucleotides. The nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II. The nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

4. The siRNA according to claim 3, wherein The length of the nucleotide sequences III and IV is 1 nucleotide, and the base of the nucleotide sequence III is A; Alternatively, the nucleotide sequences III and IV are both 2 nucleotides in length, and the base composition of the nucleotide sequence III is UA in the direction from the 5' end to the 3' end; Alternatively, the nucleotide sequences III and IV are both 3 nucleotides in length, and the base composition of the nucleotide sequence III is AUA from the 5' end to the 3' end; Alternatively, the lengths of the nucleotide sequences III and IV are both 4 nucleotides, and the base composition of the nucleotide sequence III is UAUA in the direction from the 5' end to the 3' end.

5. The siRNA according to claim 1, wherein The antisense strand further comprises a nucleotide sequence V, which is 1 to 3 nucleotides in length and is connected to the 3' end of the antisense strand to form a 3' overhang of the antisense strand; Alternatively, the nucleotide sequence V is 2 nucleotides in length; Alternatively, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides, or the nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA.

6. The siRNA according to claim 1, wherein The sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 6: 5'-CUGAAUUCCAAAAACCAAZ3-3' (SEQ ID NO: 5); 5'-Z4UUGGUUUUUGGAAUUCAGUA-3' (SEQ ID NO: 6); Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 8: 5'-UACUGAAUUCCAAAAACCAAZ3-3' (SEQ ID NO:7); 5'-Z4UUGGUUUUUGGAAUUCAGUAUA-3' (SEQ ID NO:8); Wherein, Z4 is the first nucleotide at the 5' end of the antisense strand, Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3.

7. The siRNA according to claim 6, wherein The siRNA is siPKKa1 or siPKKa2:siPKKa1 Sense strand: 5'-CUGAAUUCCAAAAACCAAU-3' (SEQ ID NO: 9) Antisense strand: 5'-AUUGGUUUUUGGAAUUCAGUA-3' (SEQ ID NO: 10) siPKKa2 Sense strand: 5'-UACUGAAUUCCAAAAACCAAU-3' (SEQ ID NO: 11) Antisense strand: 5'-AUUGGUUUUUGGAAUUCAGUAUA-3' (SEQ ID NO: 12).

8. The siRNA according to claim 1, wherein At least one nucleotide in the sense strand or the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group.

9. The siRNA according to claim 8, wherein The phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom, and the thiophosphate group is connected and exists in at least one of the group consisting of the following positions: between the first and second nucleotides at the 5' end of the sense strand; between the second and third nucleotides at the 5' end of the sense strand; between the first and second nucleotides at the 3' end of the sense strand; between the second and third nucleotides at the 3' end of the sense strand; between the first and second nucleotides at the 5' end of the antisense strand; between the second and third nucleotides at the 5' end of the antisense strand; between the first and second nucleotides at the 3' end of the antisense strand; and between the second and third nucleotides at the 3' end of the antisense strand.

10. The siRNA according to claim 1, wherein The 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

11. The siRNA according to claim 1, wherein The siRNA is siPKKa1-M1, siPKKa1-M2, siPKKa1-M3, siPKKa2-M1, siPKKa2-M2, siPKKa2-M3, siPKKa1-M1S, siPKKa1-M2S, siPKKa1-M3S, siPKKa2-M1S, siPKKa2-M2S, siPKKa2-M3S, siPKKa1-M1P 1. siPKKa1-M2P1, siPKKa1-M3P1, siPKKa2-M1P1, siPKKa2-M2P1, siPKKa2-M3P1, siPKKa1-M1S Any one of P1, siPKKa1-M2SP1, siPKKa1-M3SP1, siPKKa2-M1SP1, siPKKa2-M2SP1, siPKKa2-M3SP1, siPKKa1-M1 Sense strand: CmUmGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 13) Antisense strand: AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAm (SEQ ID NO: 14) siPKKa1-M2 Sense strand: CmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 15) Antisense strand: AmUfUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmUmAm (SEQ ID NO: 16) siPKKa1-M3 Sense strand: CmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 17) Antisense strand: AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAm (SEQ ID NO: 18) siPKKa2-M1 Sense strand: UmAmCmUmGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 19) Antisense strand: AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmUmAm (SEQ ID NO: 20) siPKKa2-M2 Sense strand: UmAmCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 21) Antisense strand: AmUfUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmUmAmUmAm (SEQ ID NO: 22) siPKKa2-M3 Sense strand: UmAmCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 23) Antisense strand: AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmUmAm (SEQ ID NO: 24) siPKKa1-M1 S is Sense strand: CmsUmsGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 25) Antisense strand: AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmsUmsAm (SEQ ID NO: 26) siPKKa1-M2S Sense strand: CmsUmsGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 27) Antisense strand: AmsUfsUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmsUmsAm (SEQ ID NO: 28) siPKKa1-M3S Sense strand: CmsUmsGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 29) Antisense strand: AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmsUmsAm (SEQ ID NO: 30) siPKKa2-M1 S is Sense strand: UmsAmsCmUmGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 31) Antisense strand: AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmsUmsAm (SEQ ID NO: 32) siPKKa2-M2S is Sense strand: UmsAmsCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 33) Antisense strand: AmsUfsUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmUmAmsUmsAm (SEQ ID NO: 34) siPKKa2-M3S is the positive strand: UmsAmsCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 35) Antisense strand: AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmsUmsAm (SEQ ID NO: 36) siPKKa1-M1P1 is the positive strand: CmUmGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 37) Antisense strand: P1AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAm (SEQ ID NO: 38) siPKKa1-M2P1 is the positive strand: CmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 39) Antisense strand: P1AmUfUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmUmAm (SEQ ID NO: 40) siPKKa1-M3P1 is the positive strand: CmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 41) Antisense strand: P1AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAm (SEQ ID NO: 42) siPKKa2-M1P1 Sense strand: UmAmCmUmGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 43) Antisense strand: P1AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmUmAm (SEQ ID NO: 44) siPKKa2-M2P1 Sense strand: UmAmCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 45) Antisense strand: P1AmUfUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmUmAmUmAm (SEQ ID NO: 46) siPKKa2-M3P1 Sense strand: UmAmCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 47) Antisense strand: P1AmUfUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmUmAm (SEQ ID NO: 48) siPKKa1-M1 SP1 Sense strand: CmsUmsGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 49) Antisense strand: P1AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmsUmsAm (SEQ ID NO: 50) siPKKa1-M2SP1 Sense strand: CmsUmsGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 51) Antisense strand: P1AmsUfsUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmsUmsAm (SEQ ID NO: 52) siPKKa1-M3SP1 Sense strand: CmsUmsGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 53) Antisense strand: P1AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmsUmsAm (SEQ ID NO: 54) siPKKa2-M1 SP1 Sense strand: UmsAmsCmUmGmAmAmUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 55) Antisense strand: P1AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmsUmsA m (SEQ ID NO: 56) siPKKa2-M2SP1 Sense strand: UmsAmsCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 57) Antisense strand: P1AmsUfsUmGmGmUfUmUfUfUmGmGmAmAfUmUfCmAmGmUmAmsUmsAm (SEQ ID NO: 58) siPKKa2-M3SP1 Sense strand: UmsAmsCmUmGmAmAfUmUfCfCfAmAmAmAmAmAmCmCmAmAmUm (SEQ ID NO: 59) Antisense strand: P1AmsUfsUmGmGmUfUmUmUmUmGmGmAmAfUmUfCmAmGmUmAmsUmsAm (SEQ ID NO: 60).

12. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the siRNA according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition according to claim 12, wherein The weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1:(1-500).

14. The pharmaceutical composition according to claim 13, wherein The weight ratio of the siRNA to the pharmaceutically acceptable carrier is 1:(1-50).

15. An siRNA conjugate, comprising the siRNA according to any one of claims 1 to 11 and a conjugated group conjugated to the siRNA.

16. The siRNA conjugate according to claim 15, wherein The conjugate group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked.

17. The siRNA conjugate according to claim 15 or 16, wherein The siRNA conjugate has a structure shown in formula (403).

18. Use of the siRNA according to any one of claims 1 to 11, the pharmaceutical composition according to any one of claims 12 to 14, and / or the siRNA conjugate according to any one of claims 15 to 17 in the preparation of a medicament for treating hereditary angioedema.

19. A kit, wherein: The kit contains the siRNA according to any one of claims 1 to 11, the pharmaceutical composition according to any one of claims 12 to 14, and / or the siRNA conjugate according to any one of claims 15 to 17.

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