Nucleic acid, composition and conjugate containing nucleic acid, preparation method and use

Abstract

Provided is an siRNA for inhibiting the gene expression of complement factor B (CFB), or a pharmaceutical composition or conjugate containing the siRNA. The siRNA contains a sense strand and an antisense strand. Each nucleotide in the siRNA is an independent modified or unmodified nucleotide. The sense strand contains a segment of nucleotide sequence I, and the nucleotide sequence I and a nucleotide sequence show in SEQ ID NO: 1 have an equal length and differ by no more than 3 bases. The antisense strand contains a nucleotide sequence II, and the nucleotide sequence II and a nucleotide sequence shown in SEQ ID NO: 2 have an equal length and differ by no more than 3 bases. The siRNA or the pharmaceutical composition or conjugate containing the siRNA can effectively treat and / or prevent diseases related to the mRNA level of the gene expression of the CFB.

Description

Nucleic acid, composition and conjugate containing the nucleic acid, preparation method and use thereof Technical Field

[0001] The present disclosure relates to a nucleic acid capable of inhibiting mRNA expression of the CFB gene, and compositions and conjugates containing the nucleic acid. The present disclosure also relates to methods for preparing and using the nucleic acids, compositions, and conjugates. Background Art

[0002] Complement factor B (CFB), primarily synthesized by stem cells and macrophages, is a key factor in the alternative complement pathway. Activation of the complement system can be divided into the classical, lectin, and alternative pathways. CFB plays a role in the low-level alternative complement pathway, in which C3 convertase cleaves C3 molecules into C3a and C3b. C3b, under the action of CFB and complement factor H, is converted into the C3 convertase C3bBb. C3bBb can in turn cleave additional C3 molecules, generating more surface-bound C3b, thereby exponentially amplifying complement activation through this positive feedback loop. Studies have shown that CFB plays a key role in the late stages of adipocyte differentiation and lipid droplet formation. It reaches other parts of the body through the circulatory system and, as a specialized protein of the alternative pathway, is involved in the development and progression of a variety of complement-related diseases, such as immune, renal, and neurological disorders. Therefore, there is a significant need in the art to develop new drugs that can effectively regulate CFB protein levels by regulating the mRNA expression of the CFB gene to prevent or treat diseases or symptoms related to CFB gene expression.

[0003] Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating diseases. Summary of the Invention

[0004] The inventors of the present disclosure have discovered that the following siRNAs provided herein can specifically inhibit the mRNA expression of the CFB gene in cells. Pharmaceutical compositions and siRNA conjugates containing the siRNAs disclosed herein can be effectively delivered to target tissues and / or cells, thereby demonstrating high drug potential in the treatment or prevention of diseases or symptoms associated with CFB gene expression. Therefore, the inventors have made the following inventions:

[0005] In one aspect, the present disclosure provides an siRNA capable of inhibiting the expression of CFB gene mRNA, wherein the siRNA 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:

[0006] 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 bases, 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 bases:

[0007] 5'-UCAAGUUGGUGUAAUCAGZ1-3' (SEQ ID NO: 1);

[0008] 5'-Z2CUGAUUACACCAACUUGA-3'(SEQ ID NO:2),

[0009] Wherein, Z1 is C, Z2 is G, the nucleotide sequence I contains the nucleotide Z3 corresponding to the position of Z1, the nucleotide sequence II contains the nucleotide Z4 corresponding to the position of Z2, and Z4 is the first nucleotide at the 5' end of the antisense strand;

[0010] Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 87 and differs by no more than 3 bases, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 88 and differs by no more than 3 bases:

[0011] 5'-GAGAUCUCUUUCCACUGCZ5-3' (SEQ ID NO:87);

[0012] 5'-Z6GCAGUGGAAAGAGAUCUC-3' (SEQ ID NO:88),

[0013] Wherein, Z5 is U, Z6 is A, the nucleotide sequence I contains the nucleotide Z7 corresponding to Z5, the nucleotide sequence II contains the nucleotide Z8 corresponding to Z6, and Z8 is the first nucleotide at the 5' end of the antisense chain.

[0014] On the other hand, the present disclosure also provides an siRNA conjugate, which contains the siRNA of the present disclosure and a conjugated group conjugated to the siRNA, wherein the conjugated group comprises a linker and a pharmaceutically acceptable targeting group, and the siRNA, the linker and the targeting group are covalently or non-covalently linked in sequence, and each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor.

[0015] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA and / or the siRNA conjugate of the present disclosure and a pharmaceutically acceptable carrier.

[0016] In another aspect, the present disclosure also provides a use of one or more of the siRNA, pharmaceutical composition and siRNA conjugate of the present disclosure in the preparation of a medicament for treating and / or preventing a disease or symptom associated with the mRNA level of CFB gene expression.

[0017] In yet another aspect, the present disclosure further provides a method for treating and / or preventing diseases or symptoms associated with the mRNA level of CFB gene expression, the method comprising administering one or more of the siRNA, pharmaceutical composition, and siRNA conjugate of the present disclosure to a subject in need thereof.

[0018] In yet another aspect, the present disclosure further provides a method for inhibiting the expression level of a CFB gene in a cell, comprising contacting an effective amount of one or more of the siRNA, pharmaceutical composition, and siRNA conjugate of the present disclosure with the cell.

[0019] In yet another aspect, the present disclosure further provides a kit comprising one or more of the siRNA, pharmaceutical composition, and siRNA conjugate of the present disclosure.

[0020] Incorporated by reference

[0021] 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. Beneficial effects

[0022] The siRNA, pharmaceutical composition and siRNA conjugate disclosed herein have high CFB mRNA inhibitory activity and / or can significantly treat and alleviate diseases or symptoms associated with the mRNA level of CFB gene expression.

[0023] First, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure exhibited excellent target mRNA expression inhibition activity in in vitro cell experiments. For example, in HepG2 cells in vitro, at a concentration of 50 nM, the conjugates disclosed herein exhibited inhibition rates of 77% and 79.5% against CFB mRNA, respectively. For another example, at a concentration of 5 nM, the conjugates disclosed herein also achieved an inhibition rate of 73.5%. At a concentration as low as 0.5 nM, the inhibition rate still exceeded 50%, reaching 57.5%. Therefore, the siRNA conjugates disclosed herein exhibited excellent CFB mRNA inhibition effects in vitro.

[0024] Second, the siRNA, pharmaceutical composition or siRNA conjugate provided by the present disclosure may have high stability in vivo.

[0025] Third, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure can have excellent target mRNA inhibition activity in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure exhibits an mRNA inhibition rate of at least 50%, 60%, 70%, 80%, 90%, or 95% of CFB gene expression in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure exhibits an mRNA inhibition rate of at least 50%, 60%, 70%, 80%, 90%, or 95% of CFB gene expression in an animal model in vivo. In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate provided by the present disclosure exhibits an mRNA inhibition rate of at least 50%, 60%, 70%, 80%, 90%, or 95% of CFB gene expression in vivo.

[0026] In some embodiments, the siRNA, pharmaceutical composition, or siRNA conjugate provided herein does not exhibit significant off-target effects. Off-target effects can be, for example, mRNA that inhibits the normal expression of non-target genes.

[0027] This shows that the siRNA, pharmaceutical composition, and siRNA conjugate provided by the present disclosure can inhibit the mRNA expressed by the CFB gene, effectively treat and / or prevent diseases or symptoms related to the mRNA level of the CFB gene expression, and have good application prospects.

[0028] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is a bar graph showing the relative expression levels of CFB mRNA in HepG2 cells after transfection with different concentrations of Conjugate 1 or Conjugate 2 of the present disclosure. DETAILED DESCRIPTION

[0030] 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.

[0031] definition

[0032] In the present disclosure, CFB mRNA refers to the sequence shown in Genbank Accession No. NM_001710.6. Further, unless otherwise specified, the term "target gene" used in the present disclosure refers to the CFB gene encoding the above-mentioned CFB mRNA, and the term "target mRNA" refers to the above-mentioned CFB mRNA.

[0033] In the above and below, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of letter f is a fluorine-modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of letter s are connected by a phosphorothioate group; P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analogue-modified nucleotide. In some embodiments, P1 is VP, Ps, or P representing a specific modification, wherein the letter combination VP represents 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 represents that the nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate-modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.

[0034] 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.

[0035] 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 strand are paired with bases on the other strand 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 strand is always paired with thymine (or uracil) on the other strand, and guanine is always paired with cytosine, the two strands are considered to be complementary to each other, and the sequence of the strand can be inferred from the sequence of its complementary strand. 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.

[0036] 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.

[0037] As used above and below, a "base difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the type of nucleotide at the same position in the former compared to the latter. For example, if a nucleotide in the latter is an A, and the corresponding nucleotide at the same position in the former is a U, C, G, or T, the two nucleotide sequences are considered to have a base difference at that position. In some embodiments, a base difference may also be considered to occur when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent.

[0038] In the above and below, particularly when describing the preparation method of siRNA of the present disclosure, pharmaceutical composition or siRNA conjugate, unless otherwise stated, described nucleoside monomer (nucleoside monomer) refers to, according to the kind and order of nucleotide in the siRNA for preparation or the siRNA conjugate, the modification used in phosphoramidite solid phase synthesis or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, RNA phosphoramidites is also referred to as Nucleoside phosphoramidites sometimes).Phosphoramidite solid phase synthesis is the method used in RNA synthesis known to those skilled in the art.The nucleoside monomer used in the disclosure all can be commercially obtained.

[0039] In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the covalent bonding of two or more chemical moieties, each with a specific function, to one another; accordingly, "conjugate" refers to a compound formed by covalent bonding of the chemical moieties. Furthermore, "siRNA conjugate" refers to a compound formed by covalent bonding of one or more chemical moieties with a specific function to siRNA. siRNA conjugate should be understood as a general term for multiple siRNA conjugates or an siRNA conjugate represented by a certain chemical formula, 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.

[0040] In the above or below, "substituted" or "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, "substituted" or "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 the alkoxy group are replaced by substituents. 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-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-C10 Alkylphenyl), -CN, -NO2, -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 C1-C3 alkyl, C6-C8 aryl, -OC1-C3 alkyl, -OC1-C3 alkylphenyl, halogen, -OH, -NH2, -CN or -NO2. 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.

[0041] 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.

[0042] 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.

[0043] As used herein, "alkynyl" refers to an unsaturated branched or straight chain hydrocarbon radical 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 has 2 to 20 carbon atoms, and in other embodiments, has 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.

[0044] 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.

[0045] 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.

[0046] "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, comprising 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., comprising a cyclic delocalized (4n+2)π-electron system according to Hückel theory. Heteroaryl groups include fused or bridged ring systems. In some embodiments, the heteroatom in the heteroaryl group is an oxidized heteroatom. In some embodiments, the heteroaryl group comprises one or more nitrogen atoms. In some embodiments, one or more of the nitrogen atoms in the heteroaryl group is a quaternized nitrogen atom. The heteroaryl group is attached to the remainder of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to, azacycloheptatrienyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzo[b][1,4] naphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5 ]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, furano[3,2-c]pyridinyl, 5,6,7,8 ,9,10-hexahydrocyclooctanol[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, dihydroindolinyl, isoindolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl (5,8-methano-5,6,7,8-tetrahydroquinazolinyl),8-tetrahydroquinazolinyl), naphthyl, 1,6-naphthyl-onyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, ]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl / thienyl. ,

[0047] As used herein, "halogen substituent" or "halogen" refers to fluoro, chloro, bromo and iodo, and the term "halogen" includes fluoro, chloro, bromo and iodine.

[0048] 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.

[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, 2d ed, 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, rabbits, sheep, rats, and any type of poultry.

[0051] As used herein, "treatment" refers 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, "prevention" refers to an approach to obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," siRNA, pharmaceutical composition, or siRNA conjugate 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 been made. Therefore, reference to an siRNA or siRNA conjugate described herein, including but not limited to an siRNA conjugate represented by any structural formula described herein, is intended to encompass the sodium salt or partial sodium salt form of the siRNA or siRNA conjugate.

[0053] siRNA of the present disclosure

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

[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 contains a sense strand and an antisense strand, and the sense strand and 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 siRNA sense strand to the antisense strand is 19 / 21, 21 / 21, 21 / 23, or 23 / 25.

[0057] The siRNA disclosed herein contains a sense strand and an antisense strand, wherein each nucleotide in the siRNA is independently a modified or unmodified nucleotide, wherein the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region.

[0058] In some embodiments, the siRNA of the present disclosure can be the following first or second siRNA, each of which is described below.

[0059] The first siRNA

[0060] In some embodiments, the siRNA disclosed herein is a first siRNA, 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 bases, 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 bases:

[0061] 5'-UCAAGUUGGUGUAAUCAGZ1-3' (SEQ ID NO: 1);

[0062] 5'-Z2CUGAUUACACCAACUUGA-3'(SEQ ID NO:2),

[0063] Wherein, Z1 is C, Z2 is G, the nucleotide sequence I contains the nucleotide Z3 corresponding to Z1, the nucleotide sequence II contains the nucleotide Z4 corresponding to Z2, and Z4 is the first nucleotide at the 5' end of the antisense chain.

[0064] In the context of the present disclosure, "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 at the 3' end of nucleotide sequence 1 is the nucleotide that corresponds in position to the first nucleotide at the 3' end of SEQ ID NO: 1.

[0065] In some embodiments, the nucleotide sequence I has no more than one base difference from the nucleotide sequence shown in SEQ ID NO: 1, and / or the nucleotide sequence II has no more than one base difference from the nucleotide sequence shown in SEQ ID NO: 2.

[0066] In some embodiments, the base 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 A, C, or U. In some embodiments, Z3 is a nucleotide complementary to Z4, and siRNAs having the above base differences have a higher ability to inhibit target mRNA, and these siRNAs containing base differences are also within the scope of protection of the present disclosure.

[0067] 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 means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotides; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

[0068] In some embodiments, 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:

[0069] 5'-UCAAGUUGGUGUAAUCAGZ3-3' (SEQ ID NO: 3);

[0070] 5'-Z4CUGAUUACACCAACUUGA-3'(SEQ ID NO:4),

[0071] wherein Z3 is selected from A, U, G, or C, and Z4 is a nucleotide complementary to Z3. In some embodiments, Z3 is C, and Z4 is G.

[0072] In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 1, 2, 3 or 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, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary.

[0073] In some embodiments, the nucleotide sequence 1 is identical to SEQ ID The nucleotide sequences shown in NO:1 are equal in length and differ by no more than 3 nucleotides, and the lengths of the nucleotide sequences III and IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A. In this case, the length ratio of the sense strand to the antisense strand is 20 / 20; alternatively, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, the base composition of the nucleotide sequence III is AU, and the base composition of the nucleotide sequence IV is AU. In this case, the length ratio of the sense strand to the antisense strand is 21 / 21; alternatively, the base lengths of the nucleotide sequences III and IV are both 3 nucleotides, the base composition of the nucleotide sequence III is CAU, and the base composition of the nucleotide sequence IV is AUG. In this case, the length ratio of the sense strand to the antisense strand is 22 / 22; alternatively, the base lengths of the nucleotide sequences III and IV are both 4 nucleotides, the base composition of the nucleotide sequence III is UCAU, and the base composition of the nucleotide sequence IV is AUGA. In this case, the length ratio of the sense strand to the antisense strand is 23 / 23.

[0074] 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.

[0075] The second siRNA

[0076] In some embodiments, the siRNA of the present disclosure is a second siRNA, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 87 and differs by no more than 3 bases, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 88 and differs by no more than 3 bases:

[0077] 5'-GAGAUCUCUUUCCACUGCZ5-3' (SEQ ID NO:87);

[0078] 5'-Z6GCAGUGGAAAGAGAUCUC-3' (SEQ ID NO:88),

[0079] Wherein, Z5 is U, Z6 is A, the nucleotide sequence I contains the nucleotide Z7 corresponding to Z5, the nucleotide sequence II contains the nucleotide Z8 corresponding to Z6, and Z8 is the first nucleotide at the 5' end of the antisense chain.

[0080] In some embodiments, there is no more than 1 base difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:87, and / or there is no more than 1 base difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:88.

[0081] In some embodiments, the base difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 88 includes a difference at position Z8, and Z8 is selected from C, G, or U. In some embodiments, Z7 is a nucleotide complementary to Z8. siRNAs having the above-mentioned base differences have a higher inhibitory ability against target mRNA, and these siRNAs containing base differences are also within the scope of protection of the present disclosure.

[0082] 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 means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotides; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

[0083] In some embodiments, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 89, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 90:

[0084] 5'-GAGAUCUCUUUCCACUGCZ7-3' (SEQ ID NO:89);

[0085] 5'-Z8GCAGUGGAAAGAGAUCUC-3' (SEQ ID NO:90),

[0086] wherein Z7 is selected from A, U, G, or C, and Z8 is a nucleotide complementary to Z7. In some embodiments, Z7 is U, and Z8 is A.

[0087] In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the lengths of the nucleotide sequence III and the nucleotide sequence IV are each independently 1, 2, 3 or 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, and the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary.

[0088] In some embodiments, the nucleotide sequence I is equal to the length of the nucleotide sequence shown in SEQ ID NO: 87 and differs by no more than 3 nucleotides, and the lengths of the nucleotide sequences III and IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A, in which case the ratio of the lengths of the sense strand to the antisense strand is 20 / 20; or, the lengths of the nucleotide sequences III and IV are both 2 nucleotides, the base composition of the nucleotide III is AU, and the base composition of the nucleotide IV is AU, in which case the ratio of the lengths of the sense strand to the antisense strand is 21 / 21; or, the base composition of the nucleotide sequence III is GAU; the base composition of the nucleotide sequence IV is AUC, in which case the ratio of the lengths of the sense strand to the antisense strand is 22 / 22; or, the base composition of the nucleotide sequence III is UGAU, and the base composition of the nucleotide sequence IV is AUCA, in which case the ratio of the lengths of the sense strand to the antisense strand is 23 / 23.

[0089] 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.

[0090] The following descriptions of nucleotide sequence V, nucleotide sequence VI, and nucleotide modifications in siRNAs apply to the aforementioned siRNAs of the present disclosure, e.g., the first siRNA or the second siRNA. That is, unless otherwise specified, the following description of siRNAs should be considered to describe each of the aforementioned siRNAs of the present disclosure, e.g., the first siRNA or the second siRNA. For example, unless a specific siRNA is specified, "the siRNA further comprises nucleotide sequence V" means "the siRNA of the present disclosure, e.g., the first siRNA, the second siRNA, or the third siRNA further comprises nucleotide sequence V."

[0091] In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further comprises a nucleotide sequence V having a length of 1 to 3 nucleotides and connected to the 3' end of the antisense strand to form a 3' overhang of the antisense strand.

[0092] In some embodiments, the sense strand further comprises a nucleotide sequence VI, which is 1 to 3 nucleotides in length and is linked to the 3' end of the sense strand to form a 3' overhang of the sense strand.

[0093] In some embodiments, the siRNA provided by the present disclosure includes nucleotide sequence V, but does not include nucleotide sequence VI. Thus, the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure 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 siRNA provided by the present disclosure includes nucleotide sequence V and VI. In some embodiments, the length of nucleotide sequence V is identical or different from the length of nucleotide sequence VI. Thus, the length ratio of the siRNA sense strand and antisense strand provided by the present disclosure can be (19-26): (19-26). In some embodiments, the length of the nucleotide sequence V and / or VI is 2 nucleotides, thus, the length ratio of the sense strand and antisense strand of the siRNA provided by the present disclosure can be 19 / 21, 21 / 21, 21 / 23, 23 / 23, 23 / 25 or 25 / 25.

[0094] Each nucleotide in the nucleotide sequence V can be any nucleotide. To facilitate synthesis and save costs, in some embodiments, 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 mRNA silencing activity.

[0095] Each nucleotide in the nucleotide sequence VI can be any nucleotide. To facilitate synthesis and save synthesis costs, in some embodiments, the nucleotide sequence VI is two consecutive thymine deoxyribonucleotides (dTdT) or two consecutive uracil ribonucleotides (UU); alternatively, to improve the affinity of the siRNA sense and antisense strands, the nucleotide sequence VI is identical to the nucleotides at the corresponding position of the target mRNA. Therefore, in some embodiments, the siRNA of the present disclosure comprises nucleotide sequences V and VI, and the ratio of the length of the siRNA sense and antisense strands is 21 / 21 or 23 / 23. In this case, the siRNA of the present disclosure has better mRNA silencing activity.

[0096] 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, wherein the nucleotide sequence of the target mRNA is substantially reverse complementary or completely reverse complementary to the nucleotide sequence II, or is substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of the nucleotide sequence II and the nucleotide sequence IV.

[0097] In some embodiments, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 6:

[0098] 5'-UCAAGUUGGUGUAAUCAGZ3-3' (SEQ ID NO: 5);

[0099] 5'-Z4CUGAUUACACCAACUUGAAU-3' (SEQ ID NO: 6);

[0100] 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;

[0101] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8:

[0102] 5'-AUUCAAGUUGGUGUAAUCAGZ3-3' (SEQ ID NO:7);

[0103] 5'-Z4CUGAUUACACCAACUUGAAUGA-3' (SEQ ID NO:8);

[0104] 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;

[0105] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 91, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 92:

[0106] 5'-GAGAUCUCUUUCCACUGCZ7-3' (SEQ ID NO:91);

[0107] 5'-Z8GCAGUGGAAAGAGAUCUCAU-3' (SEQ ID NO:92);

[0108] wherein said Z8 is the first nucleotide at the 5' end of the antisense strand, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7;

[0109] Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 93, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 94:

[0110] 5'-AUGAGAUCUCUUUCCACUGCZ7-3' (SEQ ID NO:93);

[0111] 5'-Z8GCAGUGGAAAGAGAUCUCAUCA-3' (SEQ ID NO:94);

[0112] Wherein, Z8 is the first nucleotide at the 5' end of the antisense strand, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7.

[0113] In some embodiments, the siRNA described herein is siCFBa1, siCFBa2, siCFBa3, siCFBb1, siCFBb2, or siCFBb3 listed in Table 1.

[0114] As previously described, the nucleotides in the siRNAs of the present disclosure are independently modified or unmodified. In some embodiments, the nucleotides in the siRNAs of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNAs of the present disclosure are modified nucleotides. These modifications to the nucleotide groups do not significantly impair or abolish the mRNA function of the siRNAs of the present disclosure in inhibiting CFB gene expression.

[0115] 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 in which the base on the nucleotide is 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 MJ Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.

[0116] 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.

[0117] 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.

[0118] The inventors of the present disclosure surprisingly discovered that the siRNA provided by the present disclosure achieved a high balance between stability in plasma and gene silencing efficiency in animal experiments.

[0119] 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, 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 the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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), in formulas (7) to (11), Base represents a nucleic acid base:

[0124] 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.

[0125] 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'-, 4'-position 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). In formulas (12)-(14), Base represents a nucleic acid base:

[0126] 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):

[0127] In the above formulae (15) and (16), R is selected from H, OH or alkoxy (O-alkyl), and Base represents a nucleic acid base.

[0128] 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).

[0129] In the compounds of formula (17)-formula (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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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;

[0134] 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;

[0135] 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.

[0136] The modified siRNA can make it harder for ribonucleases in the blood to cleave nucleic acids, thereby increasing their stability and making them more resistant to nuclease hydrolysis. Furthermore, the modified siRNA has a higher activity in inhibiting target mRNA.

[0137] 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):

[0138] This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.

[0139] 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:

[0140] between the first and second nucleotides at the 5' end of the sense strand;

[0141] between the second and third nucleotides at the 5' end of the sense strand;

[0142] between the first and second nucleotides at the 3' end of the sense strand;

[0143] between the second and third nucleotides at the 3' end of the sense strand;

[0144] between the first and second nucleotides at the 5' end of the antisense strand;

[0145] between the second and third nucleotides at the 5' end of the antisense strand;

[0146] between the first and second nucleotides at the 3' end of the antisense strand; and

[0147] between the second and third nucleotides at the 3' end of the antisense strand.

[0148] 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.

[0149] 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:

[0150] 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:

[0151] Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.

[0152] 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).

[0153] In some embodiments, the siRNAs provided by the present disclosure are one of siCFBa1-M1, siCFBa1-M2, siCFBa1-M3, siCFBa2-M1, siCFBa2-M2, siCFBa2-M3, siCFBa3-M1, siCFBa3-M2, siCFBa3-M3, siCFBa1-M1S, siCFBa1-M2S, siCFBa1-M3S, siCFBa2-M1S, siCFBa2-M2S, siCFBa2-M3S, siCFBa3-M1S, siCFBa3-M2S, siCFBa3-M3S, siCFBa1-M1P1, siCFBa1-M2P1, siCFBa1-M3P1, siCFBa2-M1P1, siCFBa2-M2P1, siCFBa2-M3P1, siCFBa3-M1P1, siCFBa3-M2P1, siCFBa3-M3P1, siCFBa1-M1SP1, siCFBa1-M2SP1, siCFBa1-M3SP1, siCFBa2-M1SP1, siCFBa2-M2SP1, siCFBa2-M3SP1, siCFBa3-M1SP1, siCFBa3-M2SP1, siCFBa3-M3SP1, siCFBb1-M1, siCFBb1-M2, siCFBb1-M3, siCFBb2-M1, siCFBb2-M2, siCFBb2-M3, siCFBb3-M1, siCFBb3-M2, siCFBb3-M3, siCFBb1-M1S, siCFBb1-M2S, siCFBb1-M3S, siCFBb2-M1S, siCFBb2-M2S, siCFBb2-M3S, siCFBb3-M1S, siCFBb3-M2S, siCFBb3-M3S, siCFBb1-M1P1, siCFBb1-M2P1, siCFBb1-M3P1, siCFBb2-M1P1, siCFBb2-M2P1, siCFBb2-M3P1, siCFBb3-M1P1, siCFBb3-M2P1, siCFBb3-M3P1, siCFBb1-M1SP1, siCFBb1-M2SP1, siCFBb1-M3SP1, siCFBb2-M1SP1, siCFBb2-M2SP1, siCFBb2-M3SP1, siCFBb3-M1SP1, siCFBb3-M2SP1 or siCFBb3-M3SP1.

[0154] In some embodiments, in the siRNA disclosed herein, at least one of the 3rd to 6th nucleotides of the nucleotide sequence II in the direction from the 5' end to the 3' end is a stabilizing modified nucleotide, wherein the stabilizing modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose of the nucleotide is replaced by a stabilizing modifying group. Compared with the siRNA in which the nucleotide at the corresponding position is an unmodified nucleotide, the thermal stability of the siRNA containing the stabilizing modified nucleotide is increased, and the steric hindrance of the stabilizing modifying group is greater than that of the 2'-O-methyl group.

[0155] In some embodiments, in the direction from the 5' end to the 3' end, the 3rd or 5th nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide. In some embodiments, in the direction from the 5' end to the 3' end, no more than 2 nucleotides in the 3-9 nucleotides in the nucleotide sequence II are the stabilizing modified nucleotides. By limiting the number of stabilizing modified nucleotides at specific positions, the siRNA of the present invention can achieve an optimal balance between pharmaceutical activity and low off-target effects while also having excellent stability. In some embodiments, in the direction from the 5' end to the 3' end, the 3rd and / or 5th nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide. In some embodiments, in the direction from the 5' end to the 3' end, the 3rd nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide. In some embodiments, in the direction from the 5' end to the 3' end, the 5th nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.

[0156] In some embodiments, in the 5' to 3' direction, no nucleotides other than nucleotides 3-9 in the nucleotide sequence II are stabilizing modified nucleotides. If at least one of nucleotides 3-6 in the nucleotide sequence II is a stabilizing modified nucleotide, the inclusion of stabilizing modified nucleotides other than nucleotides 3-9 may significantly affect the siRNA's ability to regulate the expression level of the target sequence.

[0157] In some embodiments, "increased thermal stability of siRNA" in the context of the present disclosure refers to an increase in the thermal dissociation temperature (Tm) of the siRNA. In some embodiments, "increased thermal stability of double-stranded siRNA" refers to an increase in the Tm of the siRNA by at least 0.05°C, in some embodiments by 0.1-6°C, and in some embodiments by 0.5-4°C. In some embodiments, by comprising stabilizing modified nucleotides at specific positions, the siRNA of the present disclosure unexpectedly exhibits higher CFB mRNA inhibitory activity. In some embodiments, the siRNA of the present disclosure also exhibits lower off-target effects compared to siRNAs that do not comprise stabilizing modified nucleotides.

[0158] In some embodiments, each of the stabilizing modifying groups independently has a structure represented by -XR, wherein X is O, NR', S or SiR'2; R is one of C2-C6 alkyl, substituted C2-C6 alkyl, C6-C8 aryl, and substituted C6-C8 aryl, and each R' is independently one of H, C1-C6 alkyl, substituted C1-C6 alkyl, C6-C8 aryl, and substituted C6-C8 aryl, and the substituted C2-C6 alkyl, substituted C6-C8 aryl, or substituted C1-C6 alkyl refers to a group formed by replacing one or more hydrogen atoms in a C2-C6 alkyl, a C6-C8 aryl, or a C1-C6 alkyl group with a substituent, and the substituent is selected from one or more of the following substituents: C1-C3 alkyl, C6-C8 aryl, C1-C3 alkoxy, halogen, oxysubyl and thiosubyl. It is noted that the present disclosure is not intended to cover all modification groups that conform to the above structure, but only relates to stabilizing modification groups that can increase the thermal stability of siRNA. In some embodiments, each of the stabilizing modification groups is independently selected from one of 2'-O-methoxyethyl, 2'-O-allyl, 2'-allyl, 2'-O-2-N-methylamino-2-oxyylideneethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl and 2'-O-2,4-dinitrophenyl. In some embodiments, each of the stabilizing modification groups is 2'-O-methoxyethyl.

[0159] In some embodiments, in the siRNA disclosed herein, the third or fifth nucleotide in the nucleotide sequence II, from the 5' end to the 3' end, is a 2'-O-methoxyethyl-modified nucleotide. In some embodiments, from the 5' end to the 3' end, no more than two nucleotides from the third to the ninth nucleotides in the nucleotide sequence II are 2'-O-methoxyethyl-modified nucleotides. In some embodiments, from the 5' end to the 3' end, the second, sixth, fourteenth, and sixteenth nucleotides in the nucleotide sequence II, if not 2'-O-methoxyethyl-modified nucleotides, are 2'-fluoro-modified nucleotides. In some embodiments, all nucleotides in the nucleotide sequence II are modified nucleotides; from the 5' end to the 3' end, the second, sixth, fourteenth, and sixteenth nucleotides in the nucleotide sequence II, if not 2'-O-methoxyethyl-modified nucleotides, are 2'-fluoro-modified nucleotides, and the other nucleotides in the nucleotide sequence II are each independently one of non-fluoro-modified nucleotides.

[0160] The inventors of the present disclosure unexpectedly discovered that the siRNAs provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability, but also retain high target mRNA inhibitory activity.

[0161] 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.

[0162] Pharmaceutical composition

[0163] In one aspect, the present disclosure provides a pharmaceutical composition comprising the siRNA described above as an active ingredient and a pharmaceutically acceptable carrier.

[0164] 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), poly(L-lysine, PLL ... phosphate), PPEEA) and poly (methacrylate-N, N-dimethylaminoethyl ester) (poly (2-dimethylaminoethyl methacrylate), PDMAEMA) and one or more of their derivatives.

[0165] 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).

[0166] 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.

[0167] 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.

[0168] 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.

[0169] The osmotic pressure regulator can be, for example, sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator is such that the osmotic pressure of the pharmaceutical composition is 200-700 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. In some embodiments, the dosage of the preparation made from the pharmaceutical composition during administration may be adjusted due to different administration methods.

[0170] 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, for subcutaneous, intramuscular, or intravenous administration, and can also be, but is not limited to, delivery of the pharmaceutical composition by spray administration to the lungs, or by spray administration through the lungs to other organs and tissues (such as the liver), or by oropharyngeal inhalation, or by nasal administration. In some embodiments, the pharmaceutical composition is for spray administration.

[0171] 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).

[0172] In some embodiments, the organic amine may be a compound described in Chinese patent application CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:

[0173] in:

[0174] Each X 101 or X 102 are each independently O, S, NA or CA, wherein A is hydrogen or C1-C 20 hydrocarbon chain;

[0175] Each Y 101 or Z 101Each independently is C=O, C=S, S=O, CH-OH or SO2;

[0176] 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;

[0177] x is an integer from 1 to 10;

[0178] 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;

[0179] 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):

[0180] 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).

[0181] 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.

[0182] 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):

[0183] 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 103Possible 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).

[0184] Among them, the compound represented by formula (201) can be prepared according to the description in Chinese patent application CN103380113A.

[0185] In some embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215):

[0186] The helper lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative;

[0187] The PEGylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0188] 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).

[0189] 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.

[0190] 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.

[0191] 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:

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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 100 kDa 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 on a 0.22 μm filter.

[0197] siRNA conjugates

[0198] In another aspect, the present disclosure provides an siRNA conjugate, comprising the above-mentioned siRNA and a conjugation group conjugated to the siRNA.

[0199] In general, the conjugated group includes at least one pharmaceutically acceptable targeting group, or further includes a 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 the siRNA and the 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 conjugated group is at the 3' end of the siRNA sense strand. In some embodiments, the conjugated group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugated group can also be connected to the 3'-hydroxyl group, and the nucleotides are connected using 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 in hepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.

[0200] The targeting group can be connected to the siRNA molecule via a suitable joint, and those skilled in the art can select a suitable joint according to the specific type of the targeting group. The types of these joints, targeting groups and the connection mode with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference. In some embodiments, the siRNA can be connected to the conjugated group by acid-labile or reducible chemical bonds, which are degradable in the acidic environment of the cell endosome, thereby making the siRNA a free state. For non-degradable conjugated modes, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.

[0201] In some embodiments, the 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.

[0202] In some embodiments, at least one or each of the targeting groups is selected from ligands that can bind to a surface receptor on a cell expressing the CFB gene.

[0203] In some embodiments, at least one or each of the targeting groups is selected from a ligand capable of binding to a mammalian hepatocyte surface receptor (ASGPR). In some embodiments, each of the targeting groups is independently a ligand that has affinity for an asialoglycoprotein receptor on the surface of a mammalian hepatocyte. In some embodiments, each of the targeting groups is independently an asialoglycoprotein or a sugar. In some embodiments, each of the targeting groups is independently an asialoglycoprotein, such as an asialo serum globulin (ASOR) or an asialo globulin (ASF). In some embodiments, each of the targeting groups is 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, , 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 - one of 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 heptoside ethyl ester, 2,5-anhydro-D-allosenitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. In some embodiments, at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

[0204] In some embodiments, the linker in the siRNA conjugates of the present disclosure has a structure as shown in formula (301):

[0205] Wherein, k is an integer from 1 to 3;

[0206] L AHaving 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;

[0207] 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.

[0208] 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.

[0209] In some embodiments, the siRNA conjugate provided by the present disclosure has a structure as shown in formula (305):

[0210] Wherein, Nu represents the siRNA provided by the present disclosure.

[0211] In some embodiments, the linker in the siRNA conjugates of the present disclosure has the structure shown in formula (306):

[0212] 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;

[0213] In some embodiments, the siRNA conjugates of the present disclosure have a structure as shown in formula (307):

[0214] Wherein, Nu represents the siRNA provided by the present disclosure.

[0215] In some embodiments, the siRNA conjugates disclosed herein have the structure shown in formula (308):

[0216] in,

[0217] n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;

[0218] Each m1, m2 or m3 is independently an integer selected from 2-10;

[0219] 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;

[0220] R3 has the structure shown in Formula A59:

[0221] wherein E1 is OH, SH or BH2, and Nu represents the siRNA provided by the present disclosure;

[0222] 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 Heteroarylene; and 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 10Alkyl, -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), -CON(C1-C 10 Alkyl)(C1-C 10 Alkylphenyl), -CONH(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);

[0223] Each L1 is independently a linear alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms may be 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 Heteroarylene; and wherein L1 optionally has 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), -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-C10 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);

[0224] represents the site of covalent attachment of a group;

[0225] M1 represents a targeting group, and its definition and selectable range are the same as above. 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.

[0226] The skilled artisan will appreciate 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 aforementioned substitutions and / or replacements. 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, rings resulting from replacement of carbon atoms of the linear alkylene group (e.g., heterocyclylene or heteroarylene) are counted as one atom.

[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 ligands in the conjugate is at least 2. In some embodiments, n1 + n3 ≥ 2, which can result in the number of M1 ligands being at least 3, making it easier for the M1 ligand to bind to the asialoglycoprotein receptor on the liver surface, thereby promoting the conjugate to enter the cell via endocytosis. Experiments have shown that when the number of M1 ligands is greater than 3, the increased ease of binding of the M1 ligand 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 and m3 are independently selected from integers of 2-10, the spatial positions between multiple M1 ligands can be made suitable for the binding of M1 ligands to the asialoglycoprotein receptor on the liver surface. In order to make the conjugate provided by the present disclosure simpler, easier to synthesize and / or reduce costs, in some embodiments, m1, m2 and 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 and 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 and 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] According to the siRNA conjugate provided by the present disclosure, R3 is a group of the structure shown in formula A59, wherein E1 is OH, SH or BH2. Based on the consideration of the availability of raw materials for preparation, in some embodiments, E1 is OH or SH.

[0231] In some embodiments, R2 is selected to achieve the connection with the nitrogen atom of the nitrogen-containing skeleton and A59. In the context of this disclosure, "nitrogen-containing skeleton" refers to the nitrogen-containing skeleton connected to R 10 、R 11 、R 12 、R 13 、R 14 and R 15The 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 of the present disclosure is prepared by a solid phase synthesis process, the R2 group needs to contain both a connection site connected to the N atom on the nitrogen-containing skeleton and a connection site connected to the P atom in R3. In some embodiments, the site of connection to the N atom on the nitrogen-containing skeleton in R2 forms an amide bond with the N atom, and the site of connection to the P atom on R3 forms a phosphate bond with the P atom. In some embodiments, R2 is B5, B6, B5' or B6':

[0232] in, Indicates the site of covalent attachment of a group.

[0233] The value range of q2 can be an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0234] The role of L1 is to connect the M1 ligand to the N on the nitrogen-containing backbone, providing targeting functionality for the siRNA conjugates disclosed herein. In some embodiments, L1 is selected from a combination of one or more of the groups of formulae A1-A26. In some embodiments, L1 is selected from a combination of one or more of A1, A4, A5, A6, A8, A10, A11, and A13; in some embodiments, L1 is selected from a combination of at least two of A1, A4, A8, A10, and A11; in some embodiments, L1 is selected from a combination of at least two of A1, A8, and A10.

[0235] 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.

[0236] In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is 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 ligand and the N on the nitrogen-containing skeleton, and to make the spatial position between the M1 ligands more suitable for binding of the M1 ligand to the liver surface asialoglycoprotein receptor.

[0237] In some embodiments, the siRNA conjugates of the present disclosure have 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):

[0238] wherein Nu represents the siRNA disclosed herein.

[0239] 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 the 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 sense strand of the siRNA, after the conjugate provided by the present disclosure enters the cell, when unwinding, a separate siRNA antisense strand can be released to inhibit target gene expression through the RNAi mechanism.

[0240] The P atom in formula A59 can be connected to any possible position on the nucleotide in the siRNA, for example, at the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the base of the 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 to the oxygen atom formed after dehydrogenation of the 3' hydroxyl group of the 3' terminal nucleotide of the siRNA sense strand, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl group of a nucleotide in the siRNA sense strand, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl group of the 5' terminal nucleotide of the siRNA sense strand.

[0241] The inventors of the present disclosure unexpectedly discovered that the siRNAs disclosed herein and siRNA conjugates containing these siRNAs exhibit significantly improved plasma stability, reduced off-target effects, and high CFB silencing activity. Therefore, in some embodiments, the siRNA disclosed herein can be one of the siRNAs listed in Tables 1a and 1b.

[0242] Table 1a The first siRNA sequence disclosed herein

[0243] The second NA sequence disclosed in Table 1b

[0244] 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. An underlined capital letter S indicates that the nucleotide immediately to the left of the letter S is a stabilizing modified nucleotide; in some embodiments, S represents a specific stabilizing modification such as moe, wherein the underlined combination of letters moe indicates that the nucleotide immediately to the left of the combination of letters moe is a nucleotide with a 2'-O-methoxyethyl modification. In some embodiments, each S is moe.

[0245] In the siRNA, pharmaceutical composition or siRNA conjugate disclosed herein, the siRNA or siRNA conjugate may exist in the form of a partial or complete water-soluble salt, and the water-soluble salt may be an ammonium salt or an alkali metal salt thereof. In some embodiments, the alkali metal salt may be a potassium salt or a sodium salt. In some embodiments, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond carries a negative charge, which may exist in the form of a hydroxyl group or a sulfhydryl group, and the hydrogen ions in the hydroxyl group or the sulfhydryl group may also be partially or completely replaced by cations. The cation may be any cation, such as a metal cation, an ammonium ion NH4 +, one of the organic ammonium cations. In order to improve solubility, in one embodiment, 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. In some embodiments, the water-soluble salt can be an amine salt, an alkali metal salt or an alkaline earth metal salt. In some embodiments, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts. The alkali metal salt is selected from potassium salts or sodium salts, and the alkaline earth metal salt is selected from calcium salts or magnesium salts. In some embodiments, the tertiary amine salt is a triethylamine salt, a triisopropylamine salt or an N,N-diisopropylethylamine salt. In some embodiments, the pharmaceutically acceptable salt of the conjugate is a mixture of a methylamine salt and an ammonium salt of the conjugate. In some embodiments, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond is at least partially bound to a sodium ion, and the siRNA or siRNA conjugate disclosed herein exists in the form of a sodium salt or a partial sodium salt.

[0246] 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.

[0247] The siRNA conjugates disclosed herein can be prepared using any reasonable synthetic route. For example, for a conjugate molecule comprising a targeting group and an active reactive group that can react with a phosphoramidite to form a covalent bond, the active group in the conjugate molecule can be first protected with a protective agent and then attached to a solid support. Subsequently, a phosphoramidite solid-phase synthesis method is used to connect nucleoside monomers one by one in the 3' to 5' direction according to the nucleotide type and sequence of the siRNA sense and antisense strands. The connection of each nucleoside monomer includes four steps: deprotection, coupling, capping, and oxidation or sulfurization. The sense and antisense strands of the siRNA are separated and annealed to obtain the siRNA conjugates disclosed herein.

[0248] Furthermore, the preparation of siRNA conjugates can also be carried out with reference to the disclosures in existing literature. For example, WO2019010274A1 describes in Example 1 a method for sequentially reacting a linker group having a specific structure and a targeting ligand to siRNA. The entire contents of the method are incorporated herein by reference.

[0249] Application of the disclosed siRNA, pharmaceutical composition containing the siRNA, and siRNA conjugate

[0250] In yet another aspect, the present disclosure provides the use of one or more of the siRNAs, pharmaceutical compositions, and siRNA conjugates disclosed herein in the preparation of a medicament for treating and / or preventing a disease or condition associated with the level of mRNA expressed by a CFB gene. In some embodiments, the disease associated with the level of mRNA expressed by a CFB gene is atypical hemolytic uremic syndrome, age-related macular degeneration, cardiovascular disease, tumor, immune system disease, kidney disease, or nervous system disease.

[0251] In yet another aspect, the present disclosure provides a method for treating and / or preventing diseases or symptoms associated with the level of mRNA expressed by the CFB gene, the method comprising administering one or more of the siRNA, pharmaceutical composition, and siRNA conjugate of the present disclosure to a subject in need thereof.

[0252] In some embodiments, the present disclosure also provides a method for inhibiting the expression level of CFB in a cell, comprising contacting the cell with an effective dose of one or more of the siRNA, pharmaceutical composition, and siRNA conjugate of the present disclosure.

[0253] By administering the siRNA, pharmaceutical composition and / or siRNA conjugate provided by the present disclosure to a subject in need, the purpose of preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in cells can be achieved through the mechanism of regulating gene expression.

[0254] Therefore, one or more of the siRNAs, pharmaceutical compositions, and siRNA conjugates provided herein can be used to prevent and / or treat the pathological conditions or diseases, or to prepare medicaments for preventing and / or treating the pathological conditions or diseases described herein.

[0255] As used herein, the term "administration / administration" refers to placing an siRNA, pharmaceutical composition and / or siRNA conjugate into a subject's body by a method or approach that at least partially localizes the siRNA, pharmaceutical composition and / or siRNA conjugate 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, pharmaceutical composition and / or siRNA conjugate being delivered to a specific site compared to the entire body of the subject; while systemic administration results in the siRNA, pharmaceutical composition and / or siRNA conjugate being delivered to substantially the entire body of the subject. Given that the present disclosure is intended to provide a means for preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in liver cells, in some embodiments, the administration is a method that can deliver the drug to the liver.

[0256] 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, two-monthly, three-monthly, six-monthly, or annually.

[0257] The dosage of the siRNA, pharmaceutical composition and / 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 the LD50 (lethal dose that causes 50% of the population to die) and the ED50 (in quantitative reactions, the dose that can cause 50% of the maximum reaction intensity, in qualitative reactions, the dose that causes 50% of the experimental subjects to have a positive reaction). The range of human dosage can be derived based on the data obtained from cell culture analysis and animal studies.

[0258] When administering the siRNA, pharmaceutical composition and / or siRNA conjugates described herein, for example, for male or female, 6-12 week old, 18-25 g C57BL / 6J or C3H / HeNCrlVr mice, based on the amount of siRNA in the siRNA, pharmaceutical composition and / or siRNA conjugate: for siRNA conjugates formed by siRNA and pharmaceutically acceptable conjugate molecules, the amount of siRNA can be 0.001-100 mg / kg body weight, in some embodiments 0.01-50 mg / kg body weight, in further embodiments 0.05-20 mg / kg body weight, in further embodiments 0.1-15 mg / kg body weight, and in yet further embodiments 0.1-10 mg / kg body weight. When administering the siRNA, pharmaceutical composition and / or siRNA conjugates described herein, the above amounts may be preferred.

[0259] In addition, by introducing the siRNA, pharmaceutical composition and / or siRNA conjugate disclosed herein into cells, the expression of specific genes in the cells can also be inhibited through the mechanism of RNAi. In some embodiments, the cells are hepatocytes. In some embodiments, the hepatocytes can be cells selected from hepatoma cell lines such as Hep3B, HepG2, Huh7, or isolated primary hepatocytes, and in some embodiments, are primary hepatocytes.

[0260] Adopt the method that the present disclosure provides to suppress specific gene expression in cell, the dosage of the siRNA in the siRNA that provides, pharmaceutical composition and / or siRNA conjugate is that those skilled in the art easily determine according to the effect that expects to obtain.For example, in some embodiments, the siRNA dosage in the siRNA conjugate that provides is such amount: it is enough to reduce the expression of target gene, and causes 1pM to 1 μ M or 0.01nM to 100nM or 0.05nM to 50nM or to the extracellular concentration of about 5nM at the target cell surface.The amount required for reaching this local concentration will vary with various factors, and the factors include delivery method, delivery site, the number of the cell layer between the delivery site and the target cell or tissue, whether delivery is local or systemic etc.The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.

[0261] Reagent test kit

[0262] The present disclosure provides a kit comprising an effective amount of one or more of the siRNA, pharmaceutical composition, and siRNA conjugate provided by the present disclosure.

[0263] In some embodiments, the kits described herein may provide one or more of siRNA, pharmaceutical compositions, and siRNA conjugates in one 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 that provides one or more of siRNA, pharmaceutical compositions, and siRNA conjugates described herein. In some embodiments, the kits may include instructions for mixing one or more of siRNA, pharmaceutical compositions, and siRNA conjugates with a pharmaceutically acceptable carrier and / or excipient or other ingredients (if any).

[0264] In the kit of the present disclosure, the siRNA and pharmaceutically acceptable carrier and / or excipient and the pharmaceutical composition and / or siRNA conjugate, and / or pharmaceutically acceptable excipient can be provided in any form, such as liquid form, dry form or lyophilized form. In some embodiments, the siRNA and pharmaceutically acceptable carrier and / or excipient and the pharmaceutical composition and / or siRNA conjugate and optional pharmaceutically acceptable excipient are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0265] The present disclosure will be further illustrated below by way of examples, but the present disclosure is not limited thereby.

[0266] Example

[0267] 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)).

[0268] Preparation Example 1-2 Synthesis of siRNA Conjugates Provided by the Present Disclosure

[0269] According to the preparation method described in Preparation Example 13 of CN110959011A, conjugates 1-2 in Table 2 below were prepared, the only difference being that the sense and antisense strands of the siRNA contained in each siRNA conjugate were as shown in Table 2; the sense and antisense strands of the siRNA were synthesized according to the nucleic acid sequences of the siRNAs numbered as conjugates 1 to conjugates 2 in Table 2 below. Each siRNA conjugate 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)), and then the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass SP1ectrometry, purchased from Waters, model: LCT Premier).

[0270] The molecular weight of conjugate 1 was 7491.5 for the sense chain and 7490.4 for the measured value; the theoretical value for the antisense chain was 7079.7 for the measured value; the molecular weight of conjugate 2 was 7446.3 for the sense chain and 7445.4 for the measured value; the theoretical value for the antisense chain was 7093.8 for the measured value; the measured value was 7093.1 for the antisense chain.

[0271] The measured values ​​are consistent with the theoretical values, indicating that the synthesized conjugates 1-2 are the target designed double-stranded nucleic acid sequences. Each siRNA conjugate is a sodium salt having a structure represented by formula (403). The siRNA groups contained in the siRNA conjugates have the siRNA sequences corresponding to conjugates 1-2 in Table 2, respectively. The conjugate groups are linked to the 3'-ribose position of the 3'-terminal nucleotide of the sense strand of the siRNA group, and each siRNA conjugate is in the form of a sodium salt.

[0272] Table 2 siRNA sequences in siRNA conjugates

[0273] Among them, the capital letters C, G, U, A, and T 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.

[0274] Comparative Preparation Example 1 Synthesis of Reference siRNA Conjugate

[0275] The reference conjugate, designated as Reference Conjugate NC in Table 2 above, was prepared using the same method as in Preparation Example 1. The only difference was that the sense and antisense strands of the siRNA contained in Reference Conjugate NC were as shown in Table 2. Molecular weights were determined using liquid chromatography-mass spectrometry (LC-MS, Waters, model: LCT Premier). The measured values ​​were consistent with the theoretical values, indicating that the synthesized Reference Conjugate NC possessed the desired double-stranded nucleic acid sequence.

[0276] Experimental Example 1 Inhibitory activity of the conjugate disclosed herein in vitro

[0277] This experimental example investigated the inhibitory activity of the siRNA conjugate disclosed herein in human HepG2 cells that can normally express the CFB gene.

[0278] HepG2 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd.) were cultured at 37°C in an incubator containing 5% CO2 / 95% air using MEM medium (MACGENE) supplemented with 10% fetal bovine serum (FBS, RMBIO) and 1% ITS (Insulin, Transferrin, Selenium, MACGENE).

[0279] HepG2 cells were cultured at 1.0×10 5 Cells were seeded in a 12-well plate with 1 mL of cell solution per well. After culturing for 24 h, the culture medium in the wells was aspirated and 500 μL of Opti-MEM medium (GIBCO) was added to each well.

[0280] Conjugate 1, Conjugate 2 and Reference Conjugate NC prepared in Preparation Example 1 were prepared into working solutions of Conjugate 1, Conjugate 2 and Reference Conjugate NC with concentrations of 20 μM, 2 μM and 0.2 μM, respectively, using PBS buffer.

[0281] Prepare 1A1, 2A1, and 3A1 solutions. Each 1A1 solution contains 100 μL of Opti-MEM medium and 3 μL of a 20 μM working solution of Conjugate 1. Each 2A1 solution contains 100 μL of Opti-MEM medium and 3 μL of a 20 μM working solution of Conjugate 2. Each 3A1 solution contains 100 μL of Opti-MEM medium and 3 μL of a 20 μM working solution of the reference conjugate NC.

[0282] Prepare 1A2 and 2A2 solutions. Each 1A2 solution contains 100 μL of Opti-MEM medium and 3 μL of a 2 μM working solution of Conjugate 1. Each 2A2 solution contains 100 μL of Opti-MEM medium and 3 μL of a 2 μM working solution of Conjugate 2.

[0283] Prepare 1A3 and 2A3 solutions. Each 1A3 solution contains 100 μL of Opti-MEM medium and 3 μL of a 0.2 μM working solution of Conjugate 1. Each 2A3 solution contains 100 μL of Opti-MEM medium and 3 μL of a 0.2 μM working solution of Conjugate 2.

[0284] Prepare 1B solution, each 1B solution contains 100 μL Opti-MEM medium and 2 μL Lipofectamine TM 2000 (Invitrogen).

[0285] Mix 1 part of 1B solution with 1 part of 1A1 solution, 1 part of 1A2 or 1 part of 1A3 solution, and incubate at room temperature for 20 minutes to obtain transfection complexes 1X1, 1X2, and 1X3, respectively.

[0286] Mix 1 part of 1B solution with 1 part of 2A1 solution, 1 part of 2A2 solution or 1 part of 2A3 solution, and incubate at room temperature for 20 minutes to obtain transfection complexes 2X1, 2X2, and 2X3, respectively.

[0287] Mix 1 part of 1B solution with 1 part of 3A1 solution respectively, and incubate at room temperature for 20 minutes to obtain a transfection complex 3X1.

[0288] Mix 1 portion of 1B solution with 100 μL of Opti-MEM medium and incubate at room temperature for 20 minutes to obtain 4×1 transfection complexes.

[0289] To six culture wells (all of which are the above-mentioned culture wells containing HepG2 cells and 500 μL Opti-MEM medium, the same below), transfection complex 1X1, transfection complex 1X2, and transfection complex 1X3 were added respectively and mixed evenly. Each transfection complex was added to two culture wells, and the amount added to each well was 97 μL / well to obtain transfection mixtures with final concentrations of 50 nM, 5 nM, and 0.5 nM, respectively, which were recorded as conjugate group 1.

[0290] To six culture wells (all of which are the above-mentioned culture wells containing HepG2 cells and 500 μL Opti-MEM medium, the same below), transfection complex 2X1, transfection complex 2X2, and transfection complex 2X3 were added respectively and mixed evenly. Each transfection complex was added to two culture wells, and the amount added to each well was 97 μL / well to obtain transfection mixtures with final concentrations of 50 nM, 5 nM, and 0.5 nM, respectively, which were recorded as conjugate group 2.

[0291] To two culture wells (both containing HepG2 cells and 500 μL Opti-MEM medium, the same below), add 3×1 transfection complexes respectively and mix evenly. Each transfection complex is added to two culture wells, with an addition volume of 97 μL / well per well to obtain a transfection mixture with a final concentration of 50 nM, which is recorded as the NC group.

[0292] To two culture wells (both containing HepG2 cells and 500 μL Opti-MEM medium, the same below), 4×1 of transfection complex was added respectively, and mixed evenly. The amount added to each well was 97 μL / well, which was recorded as the blank control group.

[0293] After incubating the above-mentioned test group 1, test group 2, NC control group, and blank control group in the culture wells for 4 hours, the supernatant was aspirated from each well and 1000 μL of Opti-MEM medium was added to each well. The 12-well plate was placed in a CO2 incubator and incubated at 37°C for another 24 hours.

[0294] Total RNA was extracted from cells in each well using TRI Reagent (Sigma, Catalog No. T9424) according to the instructions.

[0295] For each well of cells, 1 μg of total RNA was taken and reverse transcription kit Goldenstar was used. TM The reagents provided by RT6cDNA Synthesis Kit (purchased from Beijing Qingke Xinye Biotechnology Co., Ltd., catalog number TSK301M) were selected from Goldenstar TM Oligo(dT) 17 As primers, 20 μL of reverse transcription reaction system was prepared according to the reverse transcription protocol in the kit instructions, and total RNA from cells in each well was reverse transcribed. Reverse transcription conditions were as follows: for each reverse transcription reaction system, the reverse transcription reaction system was incubated at 50°C for 50 minutes, then at 85°C for 5 minutes, and finally at 4°C for 30 seconds. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0296] 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 CFB and the internal reference gene GAPDH are shown in Table 3, 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 40 times to obtain product W1 containing the amplified target gene CFB 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 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 CFB and the internal reference gene GAPDH.

[0297] Table 3 Primer information

[0298] The comparative Ct (ΔΔCt) method was used to calculate the relative quantification of the target gene CFB in each test group (conjugate 1 group, conjugate 2 group, NC group). The calculation method is as follows:

[0299] ΔCt(test group) = Ct(test group target gene) – Ct(test group reference gene)

[0300] ΔCt(control group) = Ct(control group target gene) – Ct(control group internal reference gene)

[0301] ΔΔCt(test group)=ΔCt(test group)-ΔCt(control group average)

[0302] ΔΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0303] Wherein, ΔCt (control group average) is the arithmetic mean of the ΔCt (control group) values ​​of the two culture wells of the blank control group. Thus, each culture well of the test group and the blank control group corresponds to a ΔΔCt value.

[0304] The expression level of CFB mRNA in the test group was normalized based on the average value of the blank control group, and the average value of the CFB mRNA expression level in the blank control group was defined as 100%.

[0305] Relative expression level of CFB mRNA in the test group = 2-ΔΔCt (test group) × 100%

[0306] Test group CFB mRNA inhibition rate = (1-test group CFB mRNA relative expression level) × 100%

[0307] The experimental results are shown in Figure 1. Figure 1 is a bar graph showing the relative expression levels of CFB mRNA in HepG2 cells in vitro after transfection with 50nM, 5nM or 0.5nM of conjugate 1 or conjugate 2 of the present disclosure. The results in Figure 1 show that compared with the blank control group, CFB mRNA in HepG2 cells transfected with the reference conjugate NC was only slightly reduced; while at different concentrations, the siRNA conjugates of the present disclosure showed a high level of inhibition on CFB mRNA. For example, at a concentration of 50nM, the inhibition rates of conjugate 1 and conjugate 2 of the present disclosure on CFB mRNA were as high as 77% and 79.5%, respectively. For another example, at a concentration of 5nM, the inhibition rate of conjugate 2 also reached 73.5%. At a concentration as low as 0.5nM, the inhibition rate of conjugate 2 still exceeded 50%, reaching 57.5%. The inhibition rates of the conjugate 1 of the present disclosure also reached 62.5% and 35% at concentrations of 5 nM and 0.5 nM, respectively. The above results indicate that the siRNA conjugate of the present disclosure exhibits an excellent effect of inhibiting CFB mRNA in HepG2 cells in vitro.

[0308] 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.

[0309] 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.

[0310] 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 or unmodified nucleotide, wherein: The sense strand contains a nucleotide sequence I, and the antisense strand contains 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, 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 bases, 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 bases: 5'-UCAAGUUGGUGUAAUCAGZ1-3' (SEQ ID NO: 1); 5'-Z2CUGAUUACACCAACUUGA-3'(SEQ ID NO:2), Wherein, Z1 is C, Z2 is G, the nucleotide sequence I contains the nucleotide Z3 corresponding to the position of Z1, the nucleotide sequence II contains the nucleotide Z4 corresponding to the position of Z2, and Z4 is the first nucleotide at the 5' end of the antisense strand; Alternatively, the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 87 and differs by no more than 3 bases, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 88 and differs by no more than 3 bases: 5'-GAGAUCUCUUUCCACUGCZ5-3' (SEQ ID NO:87); 5'-Z6GCAGUGGAAAGAGAUCUC-3' (SEQ ID NO:88), Wherein, Z5 is U, Z6 is A, the nucleotide sequence I contains the nucleotide Z7 corresponding to Z5, the nucleotide sequence II contains the nucleotide Z8 corresponding to Z6, and Z8 is the first nucleotide at the 5' end of the antisense chain.

2. The siRNA according to claim 1, wherein There is no more than one base difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 1, and / or there is no more than one base difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2; Alternatively, there is no more than 1 base difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO: 87, and / or there is no more than 1 base difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:

88.

3. The siRNA according to claim 2, wherein The base difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes the difference at position Z4, and Z4 is selected from A, C or U; Alternatively, the base difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 88 includes the difference at position Z8, and Z8 is selected from C, G or U.

4. The siRNA according to claim 1, wherein Z3 is a nucleotide complementary to Z4; or Z7 is a nucleotide complementary to Z8.

5. The siRNA according to claim 1, wherein The nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary or completely reverse complementary; the substantially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences; the substantially reverse complementary means that there is no more than 1 base mismatch between the two nucleotide sequences; and the completely reverse complementary means that there is no mismatch between the two nucleotide sequences.

6. The siRNA according to claim 1, wherein The sense strand and antisense strand are the same or different in length, the sense strand is 19-23 nucleotides in length, the antisense strand is 19-26 nucleotides in length, and 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'-UCAAGUUGGUGUAAUCAGZ3-3' (SEQ ID NO: 3); 5'-Z4CUGAUUACACCAACUUGA-3' (SEQ ID NO: 4), wherein Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3; Alternatively, the nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 89, and the nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 90: 5'-GAGAUCUCUUUCCACUGCZ7-3' (SEQ ID NO:89); 5'-Z8GCAGUGGAAAGAGAUCUC-3' (SEQ ID NO:90), Wherein, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7.

7. The siRNA according to claim 6, wherein Z3 is C and Z4 is G; or Z7 is U and Z8 is A.

8. The siRNA according to claim 1, wherein the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV, the length of nucleotide sequence III and nucleotide sequence IV are each independently 1, 2, 3 or 4 nucleotides, the nucleotide sequence III is connected to the 5' end of nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of 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.

9. The siRNA according to claim 8, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 1 in length and differs by no more than 3 nucleotides, and the nucleotide sequences III and IV are both 1 nucleotide in length, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; or, the nucleotide sequences III and IV are both 2 nucleotides in length, the base composition of the nucleotide sequence III is AU, and the base composition of the nucleotide sequence IV is AU; or, the base length of the nucleotide sequences III and IV is both 3 nucleotides, the base composition of the nucleotide sequence III is CAU, and the base composition of the nucleotide sequence IV is AUG; or, the base length of the nucleotide sequences III and IV is both 4 nucleotides, the base composition of the nucleotide sequence III is UCAU, and the base composition of the nucleotide sequence IV is AUGA.

10. The siRNA according to claim 8, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 87 in length and differs by no more than 3 nucleotides, and the nucleotide sequences III and IV are both 1 nucleotide in length, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; or, the nucleotide sequences III and IV are both 2 nucleotides in length, the base composition of the nucleotide III is AU, and the base composition of the nucleotide IV is AU; or, the base composition of the nucleotide sequence III is GAU; the base composition of the nucleotide sequence IV is AUC; or, the base composition of the nucleotide sequence III is UGAU, and the base composition of the nucleotide sequence IV is AUCA.

11. The siRNA according to claim 1, wherein The siRNA further contains a nucleotide sequence V, which is 1-3 nucleotides in length and is connected to the 3' end of the antisense chain to form a 3' overhang of the antisense chain; and / or the sense chain further contains a nucleotide sequence VI, which is 1 to 3 nucleotides in length and is connected to the 3' end of the sense chain to form a 3' overhang of the sense chain.

12. The siRNA according to claim 11, wherein The nucleotide sequences V and VI are 2 nucleotides in length, and in the direction from the 5' end to the 3' end, the nucleotide sequences V and / or VI are 2 consecutive thymine deoxyribonucleotides and 2 consecutive uracil ribonucleotides; or the nucleotide sequence V is complementary to the nucleotides at the corresponding positions of the target mRNA, and / or the nucleotide sequence VI is identical to the nucleotides at the corresponding positions of the target mRNA.

13. The siRNA according to claim 8, 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'-UCAAGUUGGUGUAAUCAGZ3-3' (SEQ ID NO: 5); 5'-Z4CUGAUUACACCAACUUGAAU-3' (SEQ ID NO: 6); 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; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 7, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 8: 5'-AUUCAAGUUGGUGUAAUCAGZ3-3' (SEQ ID NO:7); 5'-Z4CUGAUUACACCAACUUGAAUGA-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; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 91, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 92: 5'-GAGAUCUCUUUCCACUGCZ7-3' (SEQ ID NO:91); 5'-Z8GCAGUGGAAAGAGAUCUCAU-3' (SEQ ID NO:92); wherein said Z8 is the first nucleotide at the 5' end of the antisense strand, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7; Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 93, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 94: 5'-AUGAGAUCUCUUUCCACUGCZ7-3' (SEQ ID NO:93); 5'-Z8GCAGUGGAAAGAGAUCUCAUCA-3' (SEQ ID NO:94); Wherein, Z8 is the first nucleotide at the 5' end of the antisense strand, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7.

14. The siRNA of any one of claims 1-13, wherein the siRNA is siCFBa1, siCFBa2, siCFBa3, siCFBb1, siCFBb2, or siCFBb3. 15 . The siRNA according to claim 1 , wherein at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide, and / or at least one phosphate group is a phosphate group having a modified group.

16. The siRNA of claim 1, wherein each nucleotide in the sense strand and the antisense strand is independently a fluorine-modified nucleotide or a non-fluorine-modified nucleotide.

17. The siRNA according to claim 16, wherein The fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, 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; 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.

18. The siRNA according to claim 17, wherein 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.

19. The siRNA according to claim 16, wherein The 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

20. The siRNA according to any one of claims 15-19, wherein the siRNA is one of siCFBa1-M1, siCFBa1-M2, siCFBa1-M3, siCFBa2-M1, siCFBa2-M2, siCFBa2-M3, siCFBa3-M1, siCFBa3-M2, siCFBa3-M3, siCFBa1-M1S, siCFBa1-M2S, siCFBa1-M3S, siCFBa2-M1S, siCFBa2-M2S, siCFBa2-M3S, siCFBa3-M1S, siCFBa3-M2S, siCFBa3-M3S, siCFBa1-M1P1, siCFBa1-M2P1, siCFBa1-M3P1, siCFBa2-M1P1, siCFBa2-M2P1, siCFBa2-M3P1, siCFBa3-M1P1, siCFBa3-M2P1, siCFBa3-M3P1, siCFBa1-M1SP1, siCFBa1-M2SP1, siCFBa1-M3SP1, siCFBa2-M1SP1, siCFBa2-M2SP1, siCFBa2-M3SP1, siCFBa3-M1SP1, siCFBa3-M2SP1, siCFBa3-M3SP1, siCFBb1-M1, siCFBb1-M2, siCFBb1-M3, siCFBb2-M1, siCFBb2-M2, siCFBb2-M3, siCFBb3-M1, siCFBb3-M2, siCFBb3-M3, siCFBb1-M1S, siCFBb1-M2S, siCFBb1-M3S, siCFBb2-M1S, siCFBb2-M2S, siCFBb2-M3S, siCFBb3-M1S, siCFBb3-M2S, siCFBb3-M3S, siCFBb1-M1P1, siCFBb1-M2P1, siCFBb1-M3P1, siCFBb2-M1P1, siCFBb2-M2P1, siCFBb2-M3P1, siCFBb3-M1P1, siCFBb3-M2P1, siCFBb3-M3P1, siCFBb1-M1SP1, siCFBb1-M2SP1, siCFBb1-M3SP1, siCFBb2-M1SP1, siCFBb2-M2SP1, siCFBb2-M3SP1, siCFBb3-M1SP1, siCFBb3-M2SP1 or siCFBb3-M3SP1.

21. The siRNA according to claim 16, wherein From the 5' end to the 3' end, at least one of the 3rd to 6th nucleotides of the nucleotide sequence II is a stabilizing modified nucleotide, wherein the stabilizing modified nucleotide refers to a nucleotide in which the 2'-hydroxyl group of the ribose sugar of the nucleotide is replaced by a stabilizing modifying group. Compared with the siRNA in which the nucleotide at the corresponding position is an unmodified nucleotide, the thermal stability of the siRNA containing the stabilizing modified nucleotide is increased, and the steric hindrance of the stabilizing modifying group is greater than that of the 2'-O-methyl group.

22. The siRNA of claim 21, wherein In the direction from the 5' end to the 3' end, the third or fifth nucleotide in the nucleotide sequence II is the stabilizing modified nucleotide.

23. The siRNA of claim 21, wherein In the direction from the 5' end to the 3' end, no more than 2 nucleotides among the 3rd to 9th nucleotides in the nucleotide sequence II are the stabilizing modified nucleotides.

24. The siRNA according to any one of claims 21 to 23, wherein The increased thermal stability of the siRNA refers to an increase in the Tm of the siRNA, where Tm is the double-stranded thermal dissociation temperature of the siRNA.

25. The siRNA of claim 24, wherein The increased thermal stability of the siRNA means that the Tm of the siRNA is increased by at least 0.05°C.

26. The siRNA of claim 25, wherein The increased thermal stability of the siRNA means that the Tm of the siRNA increases by 0.1-6°C.

27. The siRNA of claim 25, wherein The increased thermal stability of the siRNA means that the Tm of the siRNA increases by 0.5-4°C.

28. The siRNA of claim 21, wherein Each of the stabilizing modifying groups independently has a structure represented by -XR, wherein X is O, NR', S or SiR'2; R is one of a C2-C6 alkyl group, a substituted C2-C6 alkyl group, a C6-C8 aryl group, and a substituted C6-C8 aryl group; each R' is independently one of H, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a C6-C8 aryl group, and a substituted C6-C8 aryl group; the substituted C2-C6 alkyl group, the substituted C6-C8 aryl group or the substituted C1-C6 alkyl group refers to a group formed by replacing one or more hydrogen atoms in a C2-C6 alkyl group, a C6-C8 aryl group or a C1-C6 alkyl group with a substituent, and the substituent is selected from one or more of the following substituents: a C1-C3 alkyl group, a C6-C8 aryl group, a C1-C3 alkoxy group, a halogen, an oxysubyl group and a thiosubyl group.

29. The siRNA of claim 28, wherein Each of the stabilizing modifying groups is independently selected from one of 2'-O-methoxyethyl, 2'-O-allyl, 2'-allyl, 2'-O-2-N-methylamino-2-oxyylideneethyl, 2'-O-2-N,N-dimethylaminoethyl, 2'-O-3-aminopropyl and 2'-O-2,4-dinitrophenyl.

30. The siRNA of claim 21, wherein From the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II are 2'-fluoro modified nucleotides if they are not 2'-O-methoxyethyl modified nucleotides.

31. The siRNA of claim 30, wherein All nucleotides in the nucleotide sequence II are modified nucleotides; from the 5' end to the 3' end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II, if not 2'-O-methoxyethyl-modified nucleotides, are 2'-fluoro-modified nucleotides, and the other nucleotides in the nucleotide sequence II are each independently one of non-fluorinated modified nucleotides.

32. The siRNA of any one of claims 21 to 31, wherein The siRNAs are siCFBa1-M1S3, siCFBa1-M2S3, siCFBa1-M3S3, siCFBa2-M1S3, siCFBa2-M2S3, siCFBa2-M3S3, siCFBa3-M1S3, siCFBa3-M2S3, siCFBa3-M3S3, siCFBa1-M1SS3, siCFBa1-M2SS3, siCFBa1-M3SS3, siCFBa2-M1SS3, siCFBa2-M2SS3, siCFBa2-M3SS3, siCFBa3-M1SS3, siCFBa3-M2SS3, siCFBa3-M3SS3, siCFBa1-M1P1S3, siCFBa1-M2P1S3, siCFBa1-M3P1S3, siCFBa2-M1P1S3, siCFBa2-M2P1S3, siCFBa2-M3P1S3, siCFBa3-M1P1S3, siCFBa3-M2P1S3, siCFBa3-M3P1S3, siCFBa1-M1SP1S3, siCFBa1-M2SP1S3, siCFBa1-M3SP1S3, siCFBa2-M1SP1S3, siCFBa2-M2SP1S3, siCFBa2-M3SP1S3, siCFBa3-M1SP1S3, siCFBa3-M2SP1S3, siCFBa3-M3SP1S3, siCFBb1-M1S3, siCFBb1-M2S3, siCFBb1-M3S3, siCFBb2-M1S3, siCFBb2-M2S3, siCFBb2-M3S3, siCFBb3-M1S3, siCFBb3-M2S3, siCFBb3-M3S3, siCFBb1-M1SS3, siCFBb1-M2SS3, siCFBb1-M3SS3, siCFBb2-M1SS3, siCFBb2-M2SS3, siCFBb2-M3SS3, siCFBb3-M1SS3, siCFBb3-M2SS3, siCFBb3-M3SS3, siCFBb1-M1P1SS3, siCFBb1-M2P1SS3, siCFBb1-M3P1S3, siCFBb2-M1P1S3, siCFBb2-M2P1S3, siCFBb2-M3P1S3, siCFBb3-M1P1S3, siCFBb3-M2P1SS3, siCFBb3-M3P1S3, siCFBb1-M1SP1S3, siCFBb1-M2SP1S3, siCFBb1-M3SP1S3, siCFBb2-M1SP1S3, siCFBb2-M2SP1S3siCFBb2-M3SP1S3, siCFBb3-M1SP1S3, siCFBb3-M2SP1S3, siCFBb3-M3SP1S3, siCFBa1-M1S5, siCFBa1-M2S5, siCFBa1-M3S5, siCFBa2-M1S5, siCFBa2-M2S5, siCFBa2-M3S5, siCFBa3-M1S5, siCFBa3-M2S5, siCFBa3-M3S5, siCFBa1-M1SS5, siCFBa1-M2SS5, siCFBa1-M3SS5, siCFBa2-M1SS5, siCFBa2-M2SS 5、siCFBa2-M3SS5、siCFBa3-M1SS5、siCFBa3-M2SS5、siCFBa3-M3SS5、siCFBa1-M1P1S5、siCFBa1-M2P1S5、siCFBa1-M3P1S5、siCFBa2-M1P1S5、siCFBa2 -M2P1S5、siCFBa2-M3P1S5、siCFBa3-M1P1S5、siCFBa3-M2P1S5、siCFBa3-M3P1S5、siCFBa1-M1SP1S5、siCFBa1-M2SP1S5、siCFBa1-M3SP1S5、siCFBa2-M 1SP1S5, siCFBa2-M2SP1S5, siCFBa2-M3SP1S5, siCFBa3-M1SP1S5, siCFBa3-M2SP1S5, siCFBa3-M3SP1S5, siCFBb1-M1S5, siCFBb1-M2S5, siCFBb1-M3S5, siCFBb2-M1S5, siCFBb2-M2S5, siCFBb2-M3S5, siCFBb3-M1S5, siCFBb3-M2S5, siCFBb3-M3S5, siCFBb1-M1SS5, siCFBb1-M2SS5, siCFBb1-M3SS5, siCF Bb2-M1SS5, siCFBb2-M2SS5, siCFBb2-M3SS5, siCFBb3-M1SS5, siCFBb3-M2SS5, siCFBb3-M3SS5, siCFBb1-M1P1SS5, siCFBb1-M2P1SS5, siCFBb1-M3P1S5, siCFBb2-M1P1S5, siCFBb2-M2P1S5, siCFBb2-M3P1S5, siCFBb3-M1P1S5, siCFBb3-M2P1SS5, siCFBb3-M3P1S5, siCFBb1-M1SP1S5, siCFBb1-M2SP1S5,One of siCFBb1-M3SP1S5, siCFBb2-M1SP1S5, siCFBb2-M2SP1S5, siCFBb2-M3SP1S5, siCFBb3-M1SP1S5, siCFBb3-M2SP1S5 or siCFBb3-M3SP1S5.

33. An siRNA conjugate, comprising the siRNA of any one of claims 1-32 and a conjugated group conjugated to the siRNA, wherein the conjugated group comprises a linker and a pharmaceutically acceptable targeting group, and wherein the siRNA, the linker and the targeting group are sequentially covalently or non-covalently linked, and each of the targeting groups is selected from a ligand capable of binding to a cell surface receptor.

34. The siRNA conjugate of claim 33, wherein The siRNA conjugate is a conjugate having a structure represented by formula (403) or a water-soluble salt thereof, wherein Nu is an siRNA group formed by siRNA represented by siCFBa1-M1S and / or siCFBb1-M1S, and the conjugated group is connected to the ribose 3' position of the 3' terminal nucleotide of the sense strand of the siRNA group; Alternatively, the water-soluble salt is an amine salt or an alkali metal salt; Alternatively, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts, and the alkali metal salt is selected from potassium salts or sodium salts; Alternatively, the tertiary amine salt is a triethylamine salt, a triisopropylamine salt or an N,N-diisopropylethylamine salt; Alternatively, the pharmaceutically acceptable salt is a sodium salt or a partial sodium salt of the conjugate.

35. A pharmaceutical composition comprising the siRNA according to any one of claims 1 to 32, and / or the siRNA conjugate according to claim 33 or 34, and a pharmaceutically acceptable carrier.

36. Use of one or more of the siRNA according to any one of claims 1 to 32, the siRNA conjugate according to claim 33 or 34, and the pharmaceutical composition according to claim 35 in the preparation of a medicament for treating and / or preventing a disease or symptom associated with the mRNA level of CFB gene expression.

37. The use according to claim 36, wherein the disease associated with the mRNA level of CFB gene expression is atypical hemolytic uremic syndrome, age-related macular degeneration, cardiovascular disease, tumor, immune system disease, kidney disease or nervous system disease.

38. A method for treating and / or preventing a disease or symptom associated with the mRNA level of CFB gene expression, the method comprising administering to a subject in need thereof an effective amount of one or more of the siRNA according to any one of claims 1 to 32, the siRNA conjugate according to claim 33 or 34, and the pharmaceutical composition according to claim 35.

39. The method of claim 38, wherein The disease associated with the mRNA level of CFB gene expression is atypical hemolytic uremic disease, age-related macular degeneration, cardiovascular disease, tumor, immune system disease, kidney disease or nervous system disease.

40. A method for inhibiting the expression level of a CFB gene in a cell, the method comprising contacting the cell with an effective dose of one or more of the siRNA according to any one of claims 1 to 32, the siRNA conjugate according to claim 33 or 34, and the pharmaceutical composition according to claim 35.

41. A kit comprising one or more of the siRNA according to any one of claims 1 to 32, the siRNA conjugate according to claim 33 or 34, and the pharmaceutical composition according to claim 35.

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