dsRNA molecules for regulating MASP2 gene activity

Abstract

The present invention provides a double-stranded RNA used to suppress the expression of mannan-binding lectin-related serine protease 2 (MASP2) in cells, cells containing the nucleotide encoding the same, and a method for treating a disease or condition mediated by or related to MASP2 expression in a subject using the dsRNA or cells.

Description

[Technical Field]

[0001] This disclosure relates to the field of RNA interference. [Background technology]

[0002] The complement system consists of more than 30 plasma-bound and cell-bound proteins and plays a crucial role in innate and adaptive immunity. Complement system proteins function through various protein interactions and cleavage events in a series of enzyme cascade reactions.

[0003] The complement system is activated through three pathways: the classical pathway, the lectin pathway, and the complement-alternative pathway. The classical pathway is usually triggered by host antibodies binding to exogenous antigens, thus requiring exposure to an antigen to elicit a specific antibody response. Therefore, the classical pathway is part of the adaptive immune system. The lectin pathway and the complement-alternative pathway are part of the innate immune system.

[0004] Mannan-binding lectin-associated serine protease-2 (MASP-2) is involved in the lectin pathway of the complement system. Inhibition of MASP-2 does not interfere with the antibody-dependent classical complement activation pathway.

[0005] The gene encoding human MASP2 is located on human chromosome 1p36.22 and is called MASP2. The propeptide encoded by this gene undergoes proteolysis to produce A and B chains, which then heterodimerize to form the 76kDa mature serine protease MASP2. MASP2 cleaves complement components C2 and C4 in the complement system's lectin pathway to produce C3 convertase. The protease MASP2 also plays a role in the coagulation cascade by cleaving prothrombin to form thrombin. MASP-2 exhibits thrombin-like activity. High levels of MASP-2 are associated with an increased risk of developing venous thromboembolism in the future. Suppressing MASP2 levels prevents thrombosis. MASP2 is highly expressed in the liver. MASP2 is also present in the brain, gallbladder, heart, kidneys, and circulatory system. Alternative splicing of this gene produces a variety of transcriptional mutants, at least one of which encodes an isoform known as Map19 (present in plasma), which is formed by proteolytic treatment.

[0006] Mannose-binding lectin (mannose-binding lectin 2, MBL) induces MASP2 self-activation through conformational changes. Activated MASP2 can initiate complement activation and exert a function to kill target cells.

[0007] Precise control of the complement system is necessary to selectively target invading microorganisms and avoid autoimmune damage (Ricklin et al., Nat.Immunol. 11:785-797, 2010). Abnormalities in the regulation of complement activity or failure to reduce complement activity can lead to autoimmune diseases, where inflammatory responses and tissue destruction can become uncontrollable. Failure to properly activate MASP2 can lead to various diseases, including IgA nephropathy and arthritis.

[0008] Currently, various inhibitors for suppressing MASP-2 are being studied. Examples of antibody-class inhibitors include monoclonal antibodies targeting MASP-2 disclosed in U.S. Patent No. 9,011,860. Examples of small molecule inhibitors include small molecule inhibitors of MASP-2 disclosed in International Publication No. 2021 / 113682(A1) and International Publication No. 2022 / 096394(A1).

[0009] Therefore, there is a need in the art for compositions and methods for treating diseases, disorders, and conditions in which suppression of MASP-2 is beneficial.

[0010] Reducing MASP-2 expression using small interfering RNAs (siRNAs) based on RNA interference mechanisms represents a novel approach for treating diseases, disorders, and conditions associated with MASP-2 activity. [Overview of the Initiative] [Means for solving the problem]

[0011] This disclosure provides a novel double-stranded RNA (dsRNA) for suppressing the expression of mannan-binding lectin-related serine protease 2 (MASP2) in cells, cells, kits, and pharmaceutical compositions containing the same, and a method for treating a disease or disorder in which suppressing or reducing the expression of the mannan-binding lectin-related serine protease 2 (MASP2) gene, or reducing the expression of mannan-binding lectin-related serine protease 2 (MASP2), is beneficial, using the above-mentioned siRNA, cells, kits, or pharmaceutical compositions.

[0012] In a first embodiment, the disclosure provides a double-stranded nucleotide (dsRNA) for repressing the expression of mannan-binding lectin-related serine protease 2 (MASP2) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, and the antisense strand comprises a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs. 22 to 40. In some embodiments, the sense strand comprises a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs. 2 to 20.

[0013] In some embodiments, the sense chain and the antisense chain are each independently 17 to 27 nucleotides long, preferably 18 to 25 nucleotides long, and more preferably 19 to 21 nucleotides long. In some embodiments, the sense chain is 15 to 27 nucleotides long, preferably 17 to 25 nucleotides long, more preferably 18 to 23 nucleotides long, more preferably 19 to 21 nucleotides long, and most preferably 19 amino acids long. In some embodiments, the antisense chain is 15 to 27 nucleotides long, preferably 17 to 25 nucleotides long, more preferably 18 to 23 nucleotides long, more preferably 19 to 22 nucleotides long, and most preferably 20 or 21 amino acids long.

[0014] In some embodiments, the above dsRNA is siRNA.

[0015] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 22 to 40, and preferably, the antisense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 22 to 40.

[0016] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 2 to 20, and preferably the antisense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 2 to 20.

[0017] In some embodiments, the siRNA comprises a pair of one sense strand sequence and an antisense strand sequence from the following pairs. (1) Sense chain: Sequence ID 2, Antisense chain: Sequence ID 22; (2) Sense chain: Sequence ID 3, Antisense chain: Sequence ID 23; (3) Sense chain: Sequence ID 4, Antisense chain: Sequence ID 24; (4) Sense chain: Sequence ID 5, Antisense chain: Sequence ID 25; (5) Sense chain: Sequence ID 6, Antisense chain: Sequence ID 26; (6) Sense chain: Sequence ID 7, Antisense chain: Sequence ID 27; (7) Sense chain: Sequence ID 8, Antisense chain: Sequence ID 28; (8) Sense chain: Sequence ID 9, Antisense chain: Sequence ID 29; (9) Sense chain: Sequence ID 10, Antisense chain: Sequence ID 30; (10) Sense chain: Sequence ID 11, Antisense chain: Sequence ID 31; (11) Sense chain: Sequence ID 12, Antisense chain: Sequence ID 32; (12) Sense chain: Sequence ID 13, Antisense chain: Sequence ID 33; (13) Sense chain: Sequence ID 14, Antisense chain: Sequence ID 34; (14) Sense chain: Sequence ID 15, Antisense chain: Sequence ID 35; (15) Sense chain: Sequence ID 16, Antisense chain: Sequence ID 36; (16) Sense chain: Sequence ID 17, Antisense chain: Sequence ID 37; (17) Sense chain: Sequence ID 18, Antisense chain: Sequence ID 38; (18) Sense strand: Sequence ID 19, Antisense strand: Sequence ID 39; and (19) Sense chain: Sequence ID 20, Antisense chain: Sequence ID 40

[0018] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

[0019] In some embodiments, the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, and phosphorothioate nucleotide inter-nucleotide bond modifications.

[0020] In some embodiments, the sense strand and / or antisense strand comprises SCP-modified nucleotides.

[0021] In some embodiments, the 3' and / or 5' ends of the sense strand and / or antisense strand contain 1 to 5 phosphorothioate nucleotide interbonds, preferably 2 to 3 phosphorothioate nucleotide interbonds.

[0022] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0023] In some embodiments, the antisense strand is 21 nucleotides long, and (i) The SCP modification at position 1 (counted from the 5' end); (ii) 2'-fluoromodifications at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) 2'-O-methyl modifications at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21 (counted from the 5' end); and / or (iv) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0024] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (ii) The SCP modification at position 1 (counted from the 5' end); (iii) 2'-fluoro modification at position 14 (counted from the 5' end); (iv) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0025] In some embodiments, the sense strand is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 It holds.

[0026] In some embodiments, the sense strand is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 17 and 18, and between nucleotide positions 18 and 19. It holds.

[0027] In some embodiments, the siRNA comprises one of the antisense strand sequences selected from Table 5. In other embodiments, the siRNA comprises one of the sense strand sequences selected from Table 4. In some embodiments, the siRNA comprises one of the sense strand sequence-antisense strand sequence pairs shown in Table 6.

[0028] In some embodiments, the antisense strand is selected from any one of the modified nucleotide sequences described in SEQ ID NOs: 100 to 122. In some embodiments, the sense strand is selected from any one of the modified nucleotide sequences described in SEQ ID NOs: 80 to 98.

[0029] In some preferred embodiments, the dsRNA comprises a pair of one of the following modified sense strand sequences and a modified antisense strand sequence. Sense chain: SEQ ID NO: 80, Antisense chain: SEQ ID NO: 100; Sense chain: Sequence ID 81, Antisense chain: Sequence ID 101; Sense chain: Sequence ID 82, Antisense chain: Sequence ID 102; Sense chain: SEQ ID NO: 83, Antisense chain: SEQ ID NO: 103; Sense chain: SEQ ID NO: 84, Antisense chain: SEQ ID NO: 104; Sense chain: Sequence ID 85, Antisense chain: Sequence ID 105; Sense chain: Sequence ID 86, Antisense chain: Sequence ID 106; Sense chain: Sequence ID 87, Antisense chain: Sequence ID 107; Sense chain: Sequence ID 88, Antisense chain: Sequence ID 108; Sense chain: SEQ ID NO: 89, Antisense chain: SEQ ID NO: 109; Sense chain: SEQ ID NO: 90, Antisense chain: SEQ ID NO: 110; Sense chain: Sequence ID 91, Antisense chain: Sequence ID 111; Sense chain: Sequence ID 92, Antisense chain: Sequence ID 112; Sense chain: Sequence ID 93, Antisense chain: Sequence ID 113; Sense chain: Sequence ID 94, Antisense chain: Sequence ID 114; Sense chain: SEQ ID NO: 95, Antisense chain: SEQ ID NO: 115; Sense chain: SEQ ID NO: 96, Antisense chain: SEQ ID NO: 116; Sense chain: Sequence ID 97, Antisense chain: Sequence ID 117; and Sense chain: SEQ ID NO: 98, Antisense chain: SEQ ID NO: 118

[0030] In some embodiments, the dsRNA is further bound to a ligand moiety containing N-acetylgalactosamine, preferably the sense strand is bound to the ligand moiety, and more preferably the 3' end of the sense strand is bound to the ligand moiety.

[0031] In some embodiments, the ligand targets the asial glycoprotein receptor (ASGPR).

[0032] In some embodiments, the ligand is [ka] (In the formula, [ka] This is a GL6 having a structure that indicates a point of attachment to the sense strand (preferably the 3' end of the sense strand) of the dsRNA via a phosphate group or a phosphorothioate group.

[0033] In some embodiments, the antisense strand includes a modified nucleotide sequence selected from any one of SEQ ID NOs: 100 to 122. In some embodiments, the sense strand includes a modified nucleotide sequence selected from any one of SEQ ID NOs: 60 to 78.

[0034] In some embodiments, the dsRNA includes a pair of modified sense strand sequences and modified antisense strand sequences from among the following pairs. Sense chain: SEQ ID NO: 60, Antisense chain: SEQ ID NO: 100; Sense chain: Sequence ID 61, Antisense chain: Sequence ID 101; Sense chain: Sequence ID 62, Antisense chain: Sequence ID 102; Sense chain: SEQ ID NO: 63, Antisense chain: SEQ ID NO: 103; Sense chain: SEQ ID NO: 64, Antisense chain: SEQ ID NO: 104; Sense chain: SEQ ID NO: 65, Antisense chain: SEQ ID NO: 105; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 106; Sense chain: Sequence ID 67, Antisense chain: Sequence ID 107; Sense chain: SEQ ID NO: 68, Antisense chain: SEQ ID NO: 108; Sense chain: SEQ ID NO: 69, Antisense chain: SEQ ID NO: 109; Sense chain: Sequence ID 70, Antisense chain: Sequence ID 110; Sense chain: Sequence ID 71, Antisense chain: Sequence ID 111; Sense chain: Sequence ID 72, Antisense chain: Sequence ID 112; Sense chain: Sequence ID 73, Antisense chain: Sequence ID 113; Sense chain: Sequence ID 74, Antisense chain: Sequence ID 114; Sense chain: Sequence ID 75, Antisense chain: Sequence ID 115; Sense chain: Sequence ID 76, Antisense chain: Sequence ID 116; Sense chain: Sequence ID 77, Antisense chain: Sequence ID 117; Sense chain: Sequence ID 78, Antisense chain: Sequence ID 118; Sense chain: SEQ ID NO: 66, Antisense chain: SEQ ID NO: 119; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 120; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 121; and Sense chain: Sequence ID 66, Antisense chain: Sequence ID 122

[0035] In another embodiment, the present disclosure provides cells containing the dsRNA of the present disclosure.

[0036] In another embodiment, the Disclosure provides a pharmaceutical composition comprising the dsRNA or cells of the Disclosure and, optionally, a pharmaceutically acceptable carrier or excipient.

[0037] In another embodiment, the Disclosure provides a kit comprising the dsRNA of the Disclosure, the cells of the Disclosure, or the pharmaceutical composition of the Disclosure.

[0038] In another embodiment, the Disclosure provides a method for treating a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) in a subject is beneficial, the method comprising the step of administering the dsRNA of the Disclosure, the cells of the Disclosure, or the pharmaceutical composition of the Disclosure to the subject.

[0039] In another embodiment, the Disclosure provides a method for preventing at least one symptom in a subject suffering from a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial, the method comprising administering the subject to the dsRNA of the Disclosure, the cells of the Disclosure, or the pharmaceutical composition of the Disclosure.

[0040] In some embodiments, the diseases or disorders in which a reduction in the expression of the mannan-binding lectin-related serine protease 2 (MASP2) is beneficial are MASP2-mediated diseases or MASP2-related diseases.

[0041] In some embodiments, the above-mentioned MASP2-mediated disease or MASP2-related disease is selected from the group consisting of arthritis, IgA nephropathy, thrombotic microangiopathy, venous embolism, diabetic nephropathy, and membranous nephropathy.

[0042] In another embodiment, the Disclosure provides a method for reducing mannan-binding lectin-associated serine protease 2 (MASP2) levels in a subject, comprising the step of administering the subject to the dsRNA of the Disclosure, the cells of the Disclosure, or the pharmaceutical composition of the Disclosure. [Modes for carrying out the invention]

[0043] Embodiments of this disclosure will be described below through specific examples. Those skilled in the art will readily understand other advantages and effects of this disclosure from this specification. Furthermore, this disclosure can also be implemented or applied through various other specific embodiments. Various details in this specification can be modified or changed in various ways on a variety of viewpoints and uses, without departing from the spirit of this disclosure.

[0044] Please understand that the scope of protection of this disclosure is not limited to the following specific embodiments. Furthermore, please understand that the terms used in the embodiments of this disclosure are for the purpose of describing the specific embodiments, and not to limit the scope of protection of this disclosure.

[0045] In the specification and claims of this disclosure, the singular forms "a," "an," and "the" include the plural form unless otherwise explicitly stated in the context.

[0046] Where numerical ranges are given in embodiments, it should be understood that, unless otherwise specifically mentioned in this disclosure, the values ​​at both ends of each numerical range and any values ​​between those ends may be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. In addition to the specific methods, apparatus, and materials used in embodiments, those skilled in the art can also implement this disclosure using similar or equivalent prior art methods, apparatus, and materials, based on their understanding of the prior art and the specifications of this disclosure, and all of these are within the scope of protection of this disclosure. Embodiments of this disclosure are described in more detail below.

[0047] definition

[0048] In this specification, “double-stranded region” means a region comprising two nucleic acid strands that are antiparallel and complementary or substantially complementary to each other.

[0049] In this specification, "double-stranded RNA" or "dsRNA" refers to a ribonucleic acid molecule or a complex of ribonucleic acid molecules containing the double-stranded region as defined above. The two parts forming the double-stranded region may be two different parts of a larger RNA molecule, or they may be separate RNA molecules.

[0050] When the two parts are separate RNA molecules, dsRNA as used herein refers to small interfering RNA or short interfering RNA, and is abbreviated as siRNA.

[0051] When two parts are two distinct parts of a larger molecule, that is, when the 3' end of one part is joined to the 5' end of the other part via one or more uninterrupted nucleotides to form a double-stranded region, the uninterrupted nucleotides used in this joining are called a "hairpin loop." When two parts are covalently joined by means other than a hairpin loop to form a double-stranded region, the joining structure is called a "linker." When such dsRNA is introduced into a cell, it can be cleaved by an endribonuclease called a dicer enzyme within the cell to become siRNA.

[0052] In this specification, “siRNA” is a class of dsRNA molecules comprising a sense strand and an antisense strand that can mediate the silencing of a target RNA (e.g., mRNA, e.g., a transcript of a protein-coding gene) that is complementary or substantially complementary to the antisense strand. siRNA is generally double-stranded and comprises an antisense strand complementary to its target RNA and a sense strand complementary or substantially complementary to the antisense strand. For convenience, such mRNA is also referred to as silencing target mRNA, and such a gene is also referred to as the target gene. Typically, silencing target RNA is an endogenous gene or a pathogen gene. In addition, RNA other than mRNA (e.g., tRNA) and viral RNA can also be targeted.

[0053] In this specification, “antisense strand” means a strand in siRNA that includes a region that is completely or substantially complementary to its target sequence.

[0054] In this specification, “complementary region” refers to a region on the antisense strand that is completely or substantially complementary to its target mRNA sequence. If the complementary region is not completely complementary to its target sequence, mismatches may be located in the internal or terminal regions of the molecule. Typically, the most acceptable mismatches are located in terminal regions such as within 5, 4, 3, 2, or 1 nucleotide at the 5' and / or 3' ends. The region of the antisense strand most sensitive to mismatches is called the “seed region.” For example, in an siRNA containing a 19nt strand, position 19 (5' to 3') may tolerate several mismatches.

[0055] In this specification, “complementary” refers to the ability of a first polynucleotide to hybridize with a second polynucleotide under certain conditions, such as stringent conditions. For example, stringent conditions include 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, and 12–16 hours at 50°C or 70°C.

[0056] In this specification, a “complementary” sequence for satisfying the above requirements regarding its hybridizing ability may include, and may consist solely of, non-Watson-Crick base pairs and / or base pairs formed with non-natural and modified nucleotides. Examples of such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuation base pairs or Hoogsteen base pairs.

[0057] In this specification, a polynucleotide that is "at least partially complementary" or "substantially complementary" to messenger RNA (mRNA) means a polynucleotide that is substantially complementary to the continuous portion of the mRNA of interest (such as the mRNA encoding MASP2). For example, a polynucleotide is at least partially complementary to the mRNA encoding MASP2 if its sequence is substantially complementary to the uninterrupted portion of the MASP2 mRNA.

[0058] In this specification, “complementary,” “fully complementary,” and “substantially complementary” may be used with respect to base pairs between the sense strand and antisense strand of an siRNA, or between the antisense strand and the target sequence of an siRNA.

[0059] In this specification, “sense strand” means one strand of siRNA that contains a region substantially complementary to the antisense strand region as defined herein.

[0060] A "nucleoside" is a compound containing two components: one is a purine or pyrimidine base, and the other is ribose or deoxyribose. A "nucleotide" is a compound containing three components: one is a purine or pyrimidine base, another is ribose or deoxyribose, and the third is phosphate. An "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) that is, for example, less than 100, 200, 300, or 400 nucleotides in length.

[0061] A "base" is the fundamental building block of nucleosides, nucleotides, and nucleic acids, and is also called a "nitrogen base" because it always contains nitrogen. Unless otherwise specified, capital letters in this specification, namely A, U, T, G, and C, indicate the bases of nucleotides, representing adenine, uracil, thymine, guanine, and cytosine, respectively.

[0062] In this specification, “nucleotide overhang” means at least one unpaired nucleotide that protrudes from the double-stranded region of an siRNA. For example, a nucleotide overhang exists when the 3' end of one strand of an siRNA extends beyond the 5' end of the other strand, or vice versa. An siRNA may contain overhangs having at least one nucleotide, at least two nucleotides, at least three nucleotides, at least four nucleotides, or at least five or more nucleotides. Nucleotide overhangs may contain or be composed of nucleotides / modified nucleotides (including deoxyribonucleotides / nucleosides). One or more overhangs may be on the sense strand or the antisense strand, or a combination thereof. Overhangs having one or more nucleotides may be located at the 5' end, 3' end, or both ends of the antisense strand or sense strand of the siRNA.

[0063] "Bluish end" means that there are no unpaired nucleotides, or nucleotide overhangs, at the end of a double-stranded siRNA. "Bluish-end siRNA" is siRNA that is double-stranded throughout its entire length, meaning that there are no nucleotide overhangs at any end of the molecule.

[0064] The siRNAs of this disclosure are substantially all modified nucleotides. For example, substantially all nucleotides of the sense strand are modified nucleotides, or substantially all nucleotides of the antisense strand are modified nucleotides, or substantially all nucleotides of both the sense and antisense strands are modified nucleotides. In other embodiments of this disclosure, all nucleotides of the siRNAs of this disclosure are modified nucleotides. For example, all nucleotides of the sense strand are modified nucleotides, or all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of both the sense and antisense strands are modified nucleotides. In this specification, “substantially all nucleotides are modified” means that the majority (but not all) of the nucleotides of the siRNAs of this disclosure are modified and may contain 5, 4, 3, 2, or 1 or fewer unmodified nucleotides.

[0065] In this specification, "modified nucleotide" is not particularly limited to, but includes 2'-O-alkyl-modified nucleotides (such as 2'-O-methyl-modified nucleotides or 2'-methoxyethyl-modified nucleotides), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, debased nucleotides, reverse debased deoxyribonucleotides, nucleotides containing a phosphorothioate group, vinyl phosphate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, and morphols. This includes nonucleotides, phosphoramidates, nucleotides containing non-natural bases, cholesteryl derivatives or terminal nucleotides bonded to a bisdecylamide dodecanoate group, deoxyribonucleotides, 3'-terminal deoxythymine (dT) nucleotides, sterically restricted nucleotides, restrictive ethyl nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides (GNAs), SCP-modified nucleotides, and 2-O-(N-methylacetamide)-modified nucleotides, etc.

[0066] For example, a "2'-fluoromodified nucleotide" refers to a nucleotide in which the hydroxyl atom at the 2' position of the ribosyl group is replaced with a fluorine atom. A "2'-O-methylmodified nucleotide" refers to a nucleotide in which the 2'-hydroxyl atom of the ribosyl group is replaced with a methoxyl atom.

[0067] A "phosphorothioate-containing nucleotide" refers to a nucleotide in which one or more oxygen atoms on the phosphate group are replaced with sulfur atoms. A "phosphorothioate internucleotide bond modification" refers to a modification in which two adjacent nucleotides are joined by a phosphorothioate.

[0068] In some embodiments, the sense strand of the siRNA of the Disclosure has at least two phosphorothioate nucleotide linkage modifications at positions 1 to 5 (counted from the 5' end) and / or at least two phosphorothioate nucleotide linkage modifications at positions 1 to 5 (counted from the 3' end), and / or the antisense strand of the siRNA of the Disclosure has at least two phosphorothioate nucleotide linkage modifications at positions 1 to 5 (counted from the 5' end) and / or at least two phosphorothioate nucleotide linkage modifications at positions 1 to 5 (counted from the 3' end).

[0069] In this specification, the “ligand moiety” refers to a chemical component bound to siRNA, which can alter the distribution, targeting, or lifespan of the siRNA. In some embodiments, the ligand moiety enhances affinity for selected targets, such as molecules, cells or cell types, and compartments (compartments of cells or organs, tissues, organs, or regions of the body), compared to siRNA without the ligand moiety. In some embodiments, the ligand moiety targets the asialocrycoprotein receptor (ASGPR) on hepatocytes. When the ligand moiety binds to ASGPR, internalization by clathrin-coated vesicles is mediated. As the endosome matures, the lysosome pH decreases, promoting the dissociation of the ligand-receptor complex and releasing the siRNA. Binding of the ligand moiety targeting the asialocrycoprotein receptor (ASGPR) on hepatocytes provides efficacy and stability of the siRNA in vivo or intracellularly. This facilitates subcutaneous administration of the siRNA.

[0070] In this specification, “suppression” is used interchangeably with similar terms such as “reduction,” “silencing,” and “downward control,” and encompasses any level of suppression.

[0071] "Suppressing the expression of mannan-binding lectin-related serine protease 2 (MASP2)" refers to suppressing the expression of the MASP2 gene or a variant or mutant of the MASP2 gene. Therefore, in the context of genetically modified cells, cell populations, or living organisms, the MASP2 gene may be a wild-type MASP2 gene, a mutant MASP2 gene, or a transgenic MASP2 gene.

[0072] "Repression of MASP2 gene expression" encompasses any level of repression of the MASP2 gene, including at least partial repression of MASP2 gene expression. MASP2 gene expression can be assessed based on the level of any variable related to MASP2 gene expression (e.g., MASP2 mRNA level or MASP2 protein level) or a change in such level. This level may be assessed in a single cell or in a population of cells (e.g., including a sample derived from the subject).

[0073] Suppression can be assessed by a decrease in the absolute or relative level of one or more variables related to MASP2 expression compared to the control level. The control level may be any type of control level used in the art, such as the baseline level before administration or the level determined in subjects, cells, or samples treated with similar untreated or controlled (such as a buffer-only control or an inactive control).

[0074] A "hydroxyl protecting group" refers to a group that can prevent a hydroxyl molecule from undergoing a chemical reaction and can be removed under certain conditions to restore the hydroxyl molecule. Examples of hydroxyl protecting groups mainly include silane, acyl, or ether-based protecting groups, preferably trimethylsilyl (TMS), triethylsilyl (TES), dimethylisopropylsilyl (DMIPS), diethylisopropylsilyl (DEIPS), tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), acetyl (Ac), chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl (TFA), benzoyl, p-methoxybenzoyl, and 9-fluorenyl methoxycarboxymethylcellulose. Examples include carbonyl (Fmoc), allyloxycarbonyl (Alloc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), benzyl (Bn), p-methoxybenzyl (PMB), allyl, triphenylmethyl (Tr), di-p-methoxytrityl (DMTr), methoxymethyl (MOM), benzyloxymethyl (BOM), 2,2,2-trichloroethoxymethyl, 2-methoxyethoxymethyl (MEM), methylthiomethyl (MTM), and p-methoxybenzyloxymethyl (PMBM).

[0075] "Halo" or "halogen" refers to substitutions by fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

[0076] "C 1-6 "Haloalkyl" refers to the above "C 1-6 This refers to a case where "alkyl" is substituted with one or more halogen groups. In some embodiments, C 1-4 Haloalkyl is particularly preferred, C 1-2Haloalkyl is even more preferred. Examples of haloalkyl include, but are not particularly limited to, -CF3, -CH2F, -CHF2, -CHFCH2F, -CH2CHF2, -CF2CF3, -CCl3, -CH2Cl, -CHCl2, and 2,2,2-trifluoro-1,1-dimethyl-ethyl, etc. The haloalkyl may be substituted at any substitutable bond point with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, etc.

[0077] "C 1-6 alkylene" refers to a divalent group formed by removing another hydrogen of C 1-6 alkyl, and may or may not be substituted. In some embodiments, C 1-4 alkylene, C 2-4 alkylene, and C 1-2 alkylene are preferred. Examples of unsubstituted alkylene include, but are not particularly limited to, methylene group (-CH2-), ethylene group (-CH2CH2-), propylene group (-CH2CH2CH2-), butylene group (-CH2CH2CH2CH2-), pentylene group (-CH2CH2CH2CH2CH2-), and hexylene group (-CH2CH2CH2CH2CH2CH2-), etc. Examples of the above substituted alkylene, for example, alkylene substituted with one or more alkyl (methyl) groups include, but are not particularly limited to, substituted methylene (-CH(CH3)- and -C(CH3)2-), substituted ethylene (-CH(CH3)CH2-, -CH2CH(CH3)-, -C(CH3)2CH2-, and -CH2C(CH3)2-), substituted propylene (-CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH(CH3)-, -C(CH3)2CH2CH2-, -CH2C(CH3)2CH2-, -CH2CH2C(CH3)2-), etc.

[0078] In this specification, “to treat” and “treatment,” etc., refer to administering a drug or performing a procedure to obtain an effect. The effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it affects the partial or complete cure of the disease and / or its symptoms. In this specification, “to treat” may include treating a disease or disorder (such as cancer) in a mammal, in particular a human, and may include (a) preventing the onset of the disease or its symptoms (e.g., diseases that may be related to or cause the underlying disease) in a subject susceptible to the disease but not diagnosed with the disease; (b) suppressing the disease, i.e., preventing its progression; and (c) alleviating the disease, i.e., causing it to regress. Treatment may refer to any reference to the success of treating, improving or preventing cancer, and may include objective or subjective parameters such as disappearance; remission; reduction of symptoms or a disease state that is tolerable to the patient; delay of exacerbation or decline; or reduction of weakness in the exacerbation endpoint. Treatment or improvement of symptoms is based on one or more objective or subjective parameters, including the results of a medical examination. Accordingly, “treatment” includes administering the antibodies, compositions, or conjugates disclosed herein to prevent, delay, alleviate, halt, or suppress the onset of symptoms or conditions associated with a disease (such as cancer). “Therapeutic effect” means reducing, eliminating, or preventing a disease, its symptoms, or its side effects in a subject.

[0079] In this specification, “effective dose” means an amount sufficient to achieve treatment of a disease when administered to a subject for the treatment of that disease.

[0080] In this specification, “subject” means a mammal subject for which diagnosis, cure, or treatment is desired. “Mammal” for therapeutic purposes means any animal classified as a mammal, including humans, livestock, laboratory animals, zoo animals, sport animals, or pet animals (such as dogs, horses, cattle, cattle, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys).

[0081] I.dsRNA

[0082] This disclosure provides a double-stranded nucleotide (dsRNA) for repressing the expression of mannan-binding lectin-related serine protease 2 (MASP2) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, and the antisense strand comprises the nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs. 22 to 40.

[0083] In some embodiments, the double-stranded regions formed by the sense strand and the antisense strand are completely complementary. In other embodiments, the double-stranded regions formed by the sense strand and the antisense strand are substantially complementary and may include one, two, three, four, or five non-complementary sites.

[0084] In some specific embodiments, the sense strand includes a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 2 to 20.

[0085] In some embodiments, the sense strand and the antisense strand are each independently 17 to 27 nucleotides long, preferably 18 to 25 nucleotides long, and more preferably 19 to 21 nucleotides long.

[0086] In some embodiments, the double-stranded region has a length of 15 to 25 nucleotide pairs, preferably 16 to 23 nucleotide pairs, and more preferably 18 to 20 nucleotide pairs.

[0087] In some embodiments, the dsRNA of the Disclosure is siRNA. In other embodiments, a hairpin loop is formed between the sense strand and the antisense strand of the dsRNA of the Disclosure.

[0088] One or both of the sense strand and the antisense strand include the 3' overhang and / or 5' overhang of at least one nucleotide. For example, one or both of the sense strand and the antisense strand include the 3' overhang and / or 5' overhang of at least one nucleotide. In some preferred embodiments, the antisense strand includes the 3' overhang and / or 5' overhang of at least two nucleotides, preferably the antisense strand includes the 3' overhang and / or 5' overhang of two nucleotides. In some embodiments, the sense strand and the antisense strand are of the same length. In some embodiments, the total length of the sense strand is complementary to the total length of the antisense strand, forming a double helix, i.e., having blunt ends. In some other embodiments, the sense strand and the antisense strand are of the same length, and a portion of the sense strand is complementary to a portion of the antisense strand, i.e., both the sense strand and the antisense strand have 5' overhangs. In some embodiments, the sense strand and the antisense strand are of different lengths. In a preferred embodiment, the 5' end of the antisense strand has an overhang of at least one nucleotide, more preferably two or three nucleotides.

[0089] The dsRNAs of this disclosure include dsRNAs having a nucleotide overhang at one end (i.e., a substance having one overhang and one blunt end), or dsRNAs having nucleotide overhangs at both ends. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the sense strand includes an overhang having one or more nucleotides. For example, the 5' end of the antisense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 5' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 3' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the antisense strand includes an overhang having one or more nucleotides. For example, the 5' end of the sense strand of a dsRNA includes an overhang having one or more nucleotides, and the 3' end of the sense strand includes a blunt end. For example, the 3' end of the sense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the sense strand contains a blunt end. For example, the 5' end of the antisense strand of dsRNA contains an overhang with one or more nucleotides, and the 3' end of the antisense strand contains a blunt end. For example, the 3' end of the antisense strand of dsRNA contains an overhang with one or more nucleotides, and the 5' end of the antisense strand contains a blunt end.

[0090] In some embodiments, the antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides in any one of the nucleotide sequences described in SEQ ID NOs: 22 to 40, and preferably the antisense strand comprises a nucleotide sequence described in any one of the nucleotide sequences described in SEQ ID NOs: 22 to 40.

[0091] In some embodiments, the sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 2 to 20, and preferably the antisense strand comprises a nucleotide sequence described in any one of SEQ ID NOs: 2 to 20.

[0092] In some embodiments, the siRNA includes a pair of sense strand sequences and antisense strand sequences from one of the following pairs: (1) Sense chain: Sequence ID 2, Antisense chain: Sequence ID 22; (2) Sense chain: Sequence ID 3, Antisense chain: Sequence ID 23; (3) Sense chain: Sequence ID 4, Antisense chain: Sequence ID 24; (4) Sense chain: Sequence ID 5, Antisense chain: Sequence ID 25; (5) Sense chain: Sequence ID 6, Antisense chain: Sequence ID 26; (6) Sense chain: Sequence ID 7, Antisense chain: Sequence ID 27; (7) Sense chain: Sequence ID 8, Antisense chain: Sequence ID 28; (8) Sense chain: Sequence ID 9, Antisense chain: Sequence ID 29; (9) Sense chain: Sequence ID 10, Antisense chain: Sequence ID 30; (10) Sense chain: Sequence ID 11, Antisense chain: Sequence ID 31; (11) Sense chain: Sequence ID 12, Antisense chain: Sequence ID 32; (12) Sense chain: Sequence ID 13, Antisense chain: Sequence ID 33; (13) Sense chain: Sequence ID 14, Antisense chain: Sequence ID 34; (14) Sense chain: Sequence ID 15, Antisense chain: Sequence ID 35; (15) Sense chain: Sequence ID 16, Antisense chain: Sequence ID 36; (16) Sense chain: Sequence ID 17, Antisense chain: Sequence ID 37; (17) Sense chain: Sequence ID 18, Antisense chain: Sequence ID 38; (18) Sense strand: Sequence ID 19, Antisense strand: Sequence ID 39; and (19) Sense chain: Sequence ID 20, Antisense chain: Sequence ID 40

[0093] II. Modification of Nucleotides

[0094] In some embodiments, substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, and at least 95% of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the antisense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, and at least 95% of the nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least 80% of the nucleotides of the sense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, and at least 95% of the nucleotides of the antisense strand are modified nucleotides, and / or at least 80%, at least 85%, at least 90%, at least 92%, and at least 95% of the nucleotides of the antisense strand are modified nucleotides.

[0095] In some embodiments, all nucleotides in the sense strand are modified nucleotides, and / or all nucleotides in the antisense strand are modified nucleotides.

[0096] The nucleotide modifications described herein may be modifications to the phosphate group, ribose group, and / or base group of the nucleotide.

[0097] In some specific embodiments, the sense chain and antisense chain are independently 2'-O-alkyl-modified nucleotides (2'-O-methyl-modified nucleotides, 2'-methoxyethyl-modified nucleotides, etc.), 2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, inosine ribonucleotides, debased nucleotides, reverse debased deoxyribonucleotides, nucleotides containing a phosphorothioate group, vinyl phosphate-modified nucleotides, locked nucleotides, unlocked nucleotides, 2'-amino-modified nucleotides, 2'-C-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, morphol The nucleotide modification comprises one or more nucleotide modifications selected from the group consisting of nonucleotides, phosphoramidates, nucleotides containing non-natural bases, cholesterol derivatives or terminal nucleotides bonded to a didecanoyl lauroyl group, deoxyribonucleotides, 3'-terminal deoxythymidine (dT) nucleotides, sterically restricted nucleotides, restrictive ethyl nucleotides, 2'-hydroxy-modified nucleotides, nucleotides containing a methylphosphonate group, nucleotides containing 5'-phosphate, nucleotides containing a 5'-phosphate mimetic, glycol-modified nucleotides (GNAs), and 2-O-(N-methylacetamide)-modified nucleotides.

[0098] In some preferred embodiments, the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, SCP modified nucleotides, and phosphorothioate internucleotide bond modifications.

[0099] In some preferred embodiments, the sense strand and / or antisense strand comprises at least two 2'-fluoromodified nucleotides. In some preferred embodiments, the sense strand and / or antisense strand comprises at least eight 2'-O-methylmodified nucleotides. In some preferred embodiments, the 3' and / or 5' ends of the sense strand and / or antisense strand comprise 1 to 5 phosphorothioate groups, preferably 2 to 3 phosphorothioate groups. In some preferred embodiments, the sense strand and / or antisense strand comprises adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and / or cytosine deoxyribonucleotide. In a more preferred embodiment, the sense strand and / or antisense strand comprises thymine deoxyribonucleotide. In the most preferred embodiment, the sense strand comprises thymine deoxyribonucleotide.

[0100] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0101] In some embodiments, the antisense strand is 21 nucleotides long, and (i) The SCP modification at position 1 (counted from the 5' end); (ii) 2'-fluoromodifications at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) 2'-O-methyl modifications at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21 (counted from the 5' end); and / or (iv) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0102] In some embodiments, the antisense strand is 21 nucleotides long, and (i) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (ii) The SCP modification at position 1 (counted from the 5' end); (iii) 2'-fluoro modification at position 14 (counted from the 5' end); (iv) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (v) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 (counted from the 5' end) It holds.

[0103] In some embodiments, the sense strand is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Internucleotide links of phosphorothioate between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between positions 18 and 19 (counted from the 5' end) It holds.

[0104] In some embodiments, the sense strand is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 17 and 18, and between nucleotide positions 18 and 19. It holds.

[0105] In some embodiments, the siRNA comprises one of the antisense strand sequences selected from Table 5. In other embodiments, the siRNA comprises one of the sense strand sequences selected from Table 4. In some embodiments, the siRNA comprises one of the sense strand sequence-antisense strand sequence pairs shown in Table 6.

[0106] III. Ligand part

[0107] The dsRNAs of this disclosure are further bound to a ligand moiety containing N-acetylgalactosamine. In preferred embodiments, the sense strand of the dsRNA is bound to the ligand moiety. In some preferred embodiments, the 3' end of the sense strand is bound to the ligand moiety. In other preferred embodiments, the 5' end of the sense strand is bound to the ligand moiety.

[0108] In some embodiments, the ligand moiety includes a binding group represented by the following formula (X').

[0109] [ka]

[0110] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA;

[0111] Q is independent of H, [ka] and;

[0112] During the ceremony, L1 represents a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -and;

[0113] L2 is a chemical bond or -CH2CH2C(O)-;

[0114] L3 is a chemical bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0115] L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -and;

[0116] During the ceremony, a = 0, 1, 2, or 3;

[0117] b = 1, 2, 3, 4, or 5;

[0118] c = 1, 2, 3, 4, or 5;

[0119] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0120] L is a chemical bond, -CH2O-, or -NHC(O)-;

[0121] L' represents a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O).e -and;

[0122] During the ceremony, e is 1, 2, 3, 4, or 5;

[0123] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0124] During the ceremony, M is [ka] and;

[0125] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0126] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0127] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0128] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0129] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0130] In some embodiments, the bonding group is represented by the following formula (I').

[0131] [ka]

[0132] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA;

[0133] Q is independent of H, [ka] and;

[0134] During the ceremony, L1 represents a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -and;

[0135] L2 is a chemical bond or -CH2CH2C(O)-;

[0136] L3 is a chemical bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0137] L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -and;

[0138] During the ceremony, a = 0, 1, 2, or 3;

[0139] b = 1, 2, 3, 4, or 5;

[0140] c = 1, 2, 3, 4, or 5;

[0141] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0142] L is either -CH2O- or -NHC(O)-;

[0143] L' is a chemical bond, -C(O)NH-, or -NHC(O)-;

[0144] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0145] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0146] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0147] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0148] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0149] In some specific embodiments, During the ceremony,

[0150] Q is independent of H or [ka] and;

[0151] During the ceremony, L1 is either -CH2O- or -NHC(O)- (CH2NHC(O)). a -and;

[0152] L2 is -CH2CH2C(O)-;

[0153] L3 is -(NHCH2CH2)b -or-(NHCH2CH2CH2) b -and;

[0154] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and;

[0155] During the ceremony, a = 0, 1, 2, or 3;

[0156] b = 1, 2, 3, 4, or 5;

[0157] c = 1, 2, 3, 4, or 5;

[0158] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0159] L is -CH2O-;

[0160] L' is a chemical bond;

[0161] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0162] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0163] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0164] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0165] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0166] In some embodiments, the bonding group is represented by the following formulas (I'-1), (I'-2), or (I'-3).

[0167] [ka]

[0168] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA;

[0169] Q is [ka] and;

[0170] During the ceremony, L1 is -CH2O- or -NHC(O)-;

[0171] L2 is -CH2CH2C(O)-;

[0172] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -and;

[0173] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and;

[0174] During the ceremony, b = 1, 2, 3, 4, or 5;

[0175] c = 1, 2, 3, 4, or 5;

[0176] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0177] L is -CH2O-;

[0178] R’ is H, a hydroxyl protecting group, or a solid support, and preferably, the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4’-dimethoxytrityl;

[0179] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0180] In some specific embodiments, in the formula,

[0181] Q is independently H,

Chemical formula

[0182] In the formula, L1 is -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0183] L3 is -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0184] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0185] In the formula, a = 0, 1, 2, or 3;

[0186] b = 1, 2, 3, 4, or 5;

[0187] c = 1, 2, 3, 4, or 5;

[0188] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0189] L is -CH2O- or -NHC(O)-;

[0190] L' is a chemical bond or -C(O)NH-;

[0191] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0192] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0193] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0194] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0195] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0196] In some embodiments, the bonding group is represented by the following formula (II'-1) or formula (II'-2).

[0197] [ka]

[0198] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA;

[0199] Q is independent, [ka] and;

[0200] During the ceremony, L1 is -CH2O- or -CH2O-CH2CH2O-;

[0201] L3 is -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0202] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -;

[0203] wherein, b = 1, 2, 3, 4, or 5;

[0204] c = 1, 2, 3, 4, or 5;

[0205] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0206] L is -NHC(O)-;

[0207] L’ is a chemical bond or -C(O)NH-;

[0208] R’ is H, a hydroxyl protecting group, or a solid support, preferably, the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4’-dimethoxytrityl;

[0209] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0210] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0211] In some specific embodiments, wherein,

[0212] Q is independently H,

Chemical formula

[0213] During the ceremony, L1 is -CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -and;

[0214] L2 is a chemical bond;

[0215] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0216] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and;

[0217] During the ceremony, a = 0, 1, 2, or 3;

[0218] b = 1, 2, 3, 4, or 5;

[0219] c = 1, 2, 3, 4, or 5;

[0220] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0221] L is either -CH2O- or -NHC(O)-;

[0222] L' is a chemical bond or -C(O)NH-;

[0223] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0224] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0225] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0226] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0227] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0228] In some embodiments, the bonding group in the formula is as shown in the following formula (II'-2).

[0229] [ka]

[0230] During the ceremony, [ka] The symbol represents the point that connects to the dsRNA;

[0231] Q is independent, [ka] and;

[0232] During the ceremony, L1 is -CH2- or -C(O)-;

[0233] L3 is -(NHCH2CH2) b -and;

[0234] L4 is -(OCH2CH2) c -and;

[0235] During the ceremony, b = 1, 2, 3, 4, or 5;

[0236] c = 1, 2, 3, 4, or 5;

[0237] L is either -CH2O- or -NHC(O)-;

[0238] R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0239] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0240] In some specific embodiments, in the formula,

[0241] Q is independent of H, [ka] and;

[0242] During the ceremony, L1 represents a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)). a -and;

[0243] L2 is a chemical bond or -CH2CH2C(O)-;

[0244] L3 is a chemical bond, -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0245] L4 is -(OCH2CH2) c -,-(OCH2CH2CH2) c -,-(OCH2CH2CH2CH2) c -,-(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -and;

[0246] During the ceremony, a = 0, 1, 2, or 3;

[0247] b = 1, 2, 3, 4, or 5;

[0248] c = 1, 2, 3, 4, or 5;

[0249] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0250] L is a chemical bond, -CH2O-, or -NHC(O)-;

[0251] L' represents a chemical bond, -C(O)NH-, -NHC(O)-, or -O(CH2CH2O). e -and;

[0252] During the ceremony, e is 1, 2, 3, 4, or 5;

[0253] T is a chemical bond, -CH2-, -M-, -CH2-M-, or -C(O)-M-;

[0254] During the ceremony, M is [ka] and;

[0255] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0256] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0257] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0258] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0259] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0260] In some specific embodiments, in the formula,

[0261] T is -M-, -CH2-M-, or -C(O)-M-, During the ceremony, M is [ka] That is the case.

[0262] In some specific embodiments, in the formula,

[0263] Q is independent of H or [ka] and;

[0264] During the ceremony, L1 is either -CH2O- or -NHC(O)- (CH2NHC(O)). a -and;

[0265] L2 is -CH2CH2C(O)-;

[0266] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -and;

[0267] L4 is -(OCH2CH2) c- or -NHC(O)-(CH2) d -and;

[0268] During the ceremony, a = 0, 1, 2, or 3;

[0269] b = 1, 2, 3, 4, or 5;

[0270] c = 1, 2, 3, 4, or 5;

[0271] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0272] L is a chemical bond or -CH2O-;

[0273] L' represents a chemical bond or -O(CH2CH2O). e -and;

[0274] During the ceremony, e is 1, 2, 3, 4, or 5;

[0275] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0276] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0277] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0278] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0279] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0280] In the formula, T is as defined in the above embodiment.

[0281] In some embodiments, the bonding group is represented by the following formulas (III'-1), (III'-2), or (III'-3).

[0282] [ka]

[0283] During the ceremony, Q is [ka] and;

[0284] During the ceremony, L1 is -CH2O- or -NHC(O)-;

[0285] L2 is -CH2CH2C(O)-;

[0286] L3 is -(NHCH2CH2) b -or-(NHCH2CH2CH2) b -and;

[0287] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and;

[0288] During the ceremony, b = 1, 2, 3, 4, or 5;

[0289] c = 1, 2, 3, 4, or 5;

[0290] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0291] L is a chemical bond or -CH2O-;

[0292] During the ceremony, R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0293] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0294] In the formula, T is as defined in the above embodiment.

[0295] In some specific embodiments, in the formula,

[0296] Q is independent of H, [ka] and;

[0297] During the ceremony, L1 is -CH2-, -CH2O-, or -C(O)-;

[0298] L2 is a chemical bond;

[0299] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0300] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and;

[0301] During the ceremony, b = 1, 2, 3, 4, or 5;

[0302] c = 1, 2, 3, 4, or 5;

[0303] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0304] L is a chemical bond or -NHC(O)-;

[0305] L' is a chemical bond;

[0306] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0307] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0308] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0309] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0310] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0311] In the formula, T is as defined in the above embodiment.

[0312] In some embodiments, the bonding group in the formula is as shown in formula (IV-1) or formula (IV-2) below.

[0313] [ka]

[0314] During the ceremony,

[0315] Q is independent, [ka] and;

[0316] During the ceremony, L1 is -CH2-, -CH2O-, or -C(O)-;

[0317] L3 is -(NHCH2CH2) b -,-(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0318] L4 is -(OCH2CH2) c - or -NHC(O)-(CH2) d -and;

[0319] During the ceremony, b = 1, 2, 3, 4, or 5;

[0320] c = 1, 2, 3, 4, or 5;

[0321] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0322] L is a chemical bond or -NHC(O)-;

[0323] L' is a chemical bond;

[0324] In the formula, R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0325] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0326] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0327] In the formula, T is as defined in the above embodiment.

[0328] In some specific embodiments, in the formula,

[0329] Q is independent of H, [ka] and;

[0330] wherein, L1 is a chemical bond, -CH2-, -CH2CH2-, -C(O)-, -CH2O-, -CH2O-CH2CH2O-, or -NHC(O)-(CH2NHC(O)) a -;

[0331] L2 is a chemical bond or -CH2CH2C(O);

[0332] L3 is a chemical bond, -(NHCH2CH2) b -, -(NHCH2CH2CH2) b -, or -C(O)CH2-;

[0333] L4 is -(OCH2CH2) c -, -(OCH2CH2CH2) c -, -(OCH2CH2CH2CH2) c -, -(OCH2CH2CH2CH2CH2) c -, or -NHC(O)-(CH2) d -;

[0334] wherein, a = 0, 1, 2, or 3;

[0335] b = 1, 2, 3, 4, or 5;

[0336] c = 1, 2, 3, 4, or 5;

[0337] d = 1, 2, 3, 4, 5, 6, 7, or 8;

[0338] L is a chemical bond, -CH2O-, or -NHC(O)-;

[0339] L’ is -O(CH2CH2O) e -;

[0340] wherein, e is 1, 2, 3, 4, or 5;

[0341] T is a chemical bond, -CH2-, -C(O)-, -M-, -CH2-M-, or -C(O)-M-;

[0342] During the ceremony, M is [ka] and;

[0343] Both R1 and R2 form -CH2CH2O- or -CH2CH(R)-O-, and R3 is H;

[0344] Alternatively, both R1 and R3 are set to -C 1-2 It forms an alkylene-, and R2 is H;

[0345] During the ceremony, R is -OR', -CH2OR', or -CH2CH2OR'; where R' is H, a hydroxyl protecting group, or a solid support, preferably the hydroxyl protecting group is -C(O)CH2CH2C(O)OH or 4,4'-dimethoxytrityl;

[0346] m = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0347] n = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0348] In some preferred embodiments, the bonding group in the formula is selected from Table 1 below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0349] In some preferred embodiments, the bonding group in the formula is selected from Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0350] In some embodiments, the ligand targets the asialocryprotein receptor (ASGPR). In some embodiments, the ligand targets the asialocryprotein receptor (ASGPR) on hepatocytes.

[0351] In a preferred embodiment, the ligand has the following structure. [ka]

[0352] During the ceremony, [ka] This indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group.

[0353] In a preferred embodiment, the ligand has the following structure. [ka]

[0354] During the ceremony, [ka] This indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group.

[0355] In a preferred embodiment, the ligand has the following structure. [ka]

[0356] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA via a phosphate group or phosphorothioate group.

[0357] In a preferred embodiment, the ligand has the following structure. [ka]

[0358] During the ceremony, [ka] This indicates a point that connects to the sense strand of dsRNA via a phosphate group or phosphorothioate group.

[0359] IV. Suppression of MASP2 gene expression

[0360] The dsRNAs of the present disclosure can suppress MASP2 gene expression by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0361] Suppression of MASP2 gene expression may be indicated by a decrease in the level of mRNA expressed in a first cell or cell population (such cells may be present, for example, in a sample derived from a subject). In such cells, the MASP2 gene is transcribed, and the cells have been treated (e.g., by contacting the cells with the dsRNAs of the present disclosure, or administering the dsRNAs of the present disclosure to a subject in which the cells are present or were previously present), whereby MASP2 gene expression is suppressed as compared to a second cell or cell population (one or more control cells) that is substantially the same as the first cell or cell population but untreated.

[0362] In a preferred embodiment, suppression is evaluated by expressing the mRNA level in the treated cells as a percentage of the mRNA level in the control cells using the following formula. In some specific embodiments, 2 -△△Ct values are calculated and the difference between the experimental group and the control group is compared. Here, △△Ct = [(Ct 標的遺伝子,実験群 - Ct 参照遺伝子,実験群 ) - (Ct 標的遺伝子,コントロール群 - Ct 参照遺伝子,コントロール群 )].[[]END]]

[0363] Alternatively, the suppression of MASP2 gene expression, such as MASP2 protein expression, may be evaluated in terms of a decrease in parameters functionally related to MASP2 gene expression, such as lipid levels and cholesterol levels (LDLc levels, etc.). MASP2 gene silencing may be measured by any assay known in the field in any cell that structurally or genomically engineered expresses MASP2. The liver is the primary site of MASP2 expression, and other important expression sites include the following:

[0364] Suppression of MASP2 protein expression can be indicated by a decrease in the level of MASP2 protein expressed in cells or cell populations (e.g., the level of protein expressed in a sample derived from the target). As explained above regarding the evaluation of mRNA suppression, the suppression of protein expression levels in treated cells or cell populations may also be expressed as a percentage of the protein level in control cells or cell populations.

[0365] Examples of control cells or cell populations that can be used to evaluate the suppression of MASP2 gene expression include cells or cell populations that have not been exposed to the dsRNA of this disclosure. For example, the control cells or cell population may be derived from individual subjects (such as human or animal subjects) before they are treated with the dsRNA. V. Cell

[0366] This disclosure provides cells containing the dsRNA of this disclosure, which are transcribable within these cells.

[0367] VI. Pharmaceutical Compositions

[0368] This disclosure provides pharmaceutical compositions comprising the dsRNA or cells of this disclosure and, optionally, pharmaceutically acceptable carriers or excipients.

[0369] As used herein, “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for contact with human and animal tissues, within the bounds of appropriate medical judgment, without causing excessive toxicity, irritation, allergic reactions, or other problems or complications, and that provides a reasonable benefit-risk ratio.

[0370] As used herein, pharmaceutically acceptable carriers refer to pharmaceutical carriers that facilitate the administration of dsRNA, vectors containing the sequence encoding it, or cells to the human body, and / or promote their absorption or action. Examples include diluents, excipients (such as water), and fillers (such as starch and sucrose); binders (such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone); humectants (such as glycerin); disintegrants (such as agar, calcium carbonate, and sodium bicarbonate); absorption enhancers (such as quaternary ammonium compounds); surfactants (such as cetyl alcohol); adsorbents (such as kaolin and bentonite); and lubricants (such as talc, calcium / magnesium stearate, and polyethylene glycol). Other adjuvants such as fragrances and sweeteners may also be added to the composition.

[0371] For example, a pharmaceutical composition comprising the dsRNA, vector, or cell of the Disclosure may contain a pharmaceutically acceptable diluent or sustained-release substrate, wherein the dsRNA or vector of the Disclosure is embedded in the sustained-release substrate.

[0372] The pharmaceutical compositions of this disclosure may include a drug delivery system for delivering dsRNA, such as nanoparticles, polymers, liposomes, PEG, or a cation delivery system.

[0373] VII. Kit

[0374] This disclosure provides a kit comprising the dsRNA, cells, or pharmaceutical composition of this disclosure.

[0375] The Disclosure also provides a kit for using the dsRNA of the Disclosure and / or for carrying out the method of the Disclosure. The kit may further include one or more of the dsRNA of the Disclosure, cells, or pharmaceutical compositions, and may also include instructions for use. The instructions for use may contain instructions for suppressing MASP2 expression in cells by contacting the cells with an amount of the dsRNA of the Disclosure effective for suppressing MASP2 expression.

[0376] When the dsRNA of the Disclosure is brought into contact with cells in vitro, the Kit of the Disclosure may further include, as necessary, means for bringing the cells into contact with the dsRNA of the Disclosure (e.g., an injection device) or means for measuring the inhibitory effect of MASP2 (e.g., a device for measuring the inhibition of MASP2 mRNA or protein). Such means for measuring the inhibition of MASP2 may include a device for obtaining a sample (e.g., a plasma sample) from the subject.

[0377] When the dsRNA or pharmaceutical composition of the Disclosure, or cells into which the dsRNA has been introduced in vitro, is administered into the body, the Kit of the Disclosure may further include, as necessary, a device for administering the dsRNA, cells, or pharmaceutical composition of the Disclosure, or a device for determining a therapeutic or prophylactic dose.

[0378] VIII. Treatment Methods and Pharmaceutical Uses

[0379] This disclosure provides a method for reducing mannan-binding lectin-related serine protease 2 (MASP2) in a subject, comprising the step of administering the dsRNA, cells, or pharmaceutical composition of this disclosure to the subject.

[0380] This disclosure provides a method for treating, preventing, suppressing, or alleviating a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial, the method comprising the step of administering the dsRNA, cells, or pharmaceutical composition of this disclosure to the subject. This disclosure also provides a method for treating, preventing, suppressing, or alleviating at least one symptom in a patient having a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial.

[0381] In some embodiments, MASP2-related diseases are diseases or disorders in which a reduction in the expression of mannan-binding lectin-associated serine protease 2 (MASP2) is beneficial. In some embodiments, MASP2-related diseases include arthritis, IgA nephropathy, thrombotic microangiopathy, venous embolism, diabetic nephropathy, and membranous nephropathy.

[0382] In some embodiments, a method for reducing mannan-binding lectin-related serine protease 2 (MASP2) in a subject, a method for treating, preventing, suppressing, or alleviating a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial in a subject, or a method for treating, preventing, suppressing, or alleviating at least one symptom in a patient having a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial, comprises administering dsRNA or a pharmaceutical composition to the subject subcutaneously or intravenously. In some embodiments, the subject is a human patient.

[0383] This disclosure also relates to the dsRNA, cells, or pharmaceutical compositions of this disclosure for use in treating diseases or conditions associated with MASP2 expression in subjects.

[0384] This disclosure also relates to the use of the dsRNA, cells, or pharmaceutical compositions of this disclosure in the manufacture of a pharmaceutical for treating, preventing, suppressing, or alleviating a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial. This disclosure also relates to the use of the dsRNA, cells, or pharmaceutical compositions of this disclosure in the manufacture of a pharmaceutical for reducing mannan-binding lectin-related serine protease 2 (MASP2) in a subject. This disclosure also relates to the use of the dsRNA, cells, or pharmaceutical compositions of this disclosure in the manufacture of a pharmaceutical for treating, preventing, suppressing, or alleviating at least one symptom in a patient having a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial. The pharmaceuticals of this disclosure can be prepared as emulsions, microemulsions, microparticles, etc.

[0385] array

[0386] The RNA sequences provided in this disclosure target the human MASP2 gene (or target gene, target mRNA sequence, target sequence). The target MASP2 mRNA sequence is represented by the Genbank accession number NM_006610.3. The dsRNA sequences provided in this disclosure are shown in Table 3. [Table 3]

[0387] Tables 4 and 5 show the modified RNA sequences used in this disclosure.

[0388] The meanings of the abbreviations used in this specification are as follows:

[0389] "A," "U," "G," and "C" represent natural adenine ribonucleotide, uracil ribonucleotide, guanine ribonucleotide, and cytosine ribonucleotide, respectively.

[0390] The letter "d" indicates that the nucleotide adjacent to it on its right is a deoxyribonucleotide. For example, "dA", "dT", "dG", and "dC" represent adenine deoxyribonucleotide, thymine deoxyribonucleotide, guanine deoxyribonucleotide, and cytosine deoxyribonucleotide, respectively.

[0391] The letter "m" indicates that the nucleotide adjacent to it on its left is a 2'-OCH3 modified nucleotide. For example, "Am", "Um", "Gm", and "Cm" represent 2'-OCH3 modified A, U, G, and C, respectively.

[0392] The letter "f" indicates that the nucleotide adjacent to it on its left is a 2'-fluoro-modified nucleotide. For example, "Af", "Uf", "Gf", and "Cf" represent 2'-fluoro-modified A, U, G, and C, respectively.

[0393] The letter "s" indicates that two adjacent nucleotides are linked by a phosphorothioate bond.

[0394] The "s-" indicates that the nucleotide adjacent to its left and the ligand adjacent to its right are linked by a phosphorothioate bond.

[0395] "SCP1a-X" has the following structure: [ka] This represents a modified nucleotide X having the following characteristics: "X" represents a nucleotide selected from the group consisting of adenine, guanine, uracil, and cytosine, and "Base" is the nucleotide base.

[0396] "SCP modification" has the following structure: [ka] A modified nucleotide having a modified base is defined as follows: "Base" is independently selected from H, a modified or unmodified base, or a leaving group. Preferably, "Base" is an unmodified base selected from the group consisting of adenine bases, guanine bases, uracil bases, and cytosine bases. In other embodiments, Base is a modified base.

[0397] "VP" indicates that the nucleotide adjacent to its right is a vinyl phosphate-modified nucleotide, which is well known in this field. For example, see PCT publication numbers WO2011139702, WO2013033230, and WO2019105419.

[0398] "L96" represents a GalNAc ligand with the following structure, which is well known in this field, and in the formula, [ka] The symbol indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group. See, for example, PCT publication numbers WO2009073809 and WO2009082607. [ka]

[0399] "GL6" represents a GalNAc ligand with the following structure, in the formula, [ka] This indicates a point that connects to dsRNA via a phosphate group or phosphorothioate group. [ka] [Table 4-1] [Table 4-2] [Table 5] [Table 6-1] [Table 6-2]

[0400] The contents of this disclosure will be further explained below with reference to examples. Please understand that the following examples are illustrative and should not be construed as limiting the scope of this disclosure. [Examples]

[0401] Unless otherwise specified, the materials used in the examples are commercially available from the following suppliers.

[0402] Huh7 cell line: Purchased from Cobioer Biosciences, Cat#CBP60202;

[0403] Hep3B cell line: Purchased from Cobioer Biosciences, Cat#CBP60197;

[0404] PHH cells: Purchased from Shanghai Yiheng Technology, Cat#QYLF-HPMC;

[0405] PMH cells: Purchased from Biocytogen Pharmaceuticals (Beijing), Cat#110872;

[0406] HEK293A cell line: Purchased from Cobioer Biosciences, Cat#CBP60436;

[0407] Balb / c mice: Purchased from Vital River Laboratory Animal Technology, Cat#Balb / c.

[0408] Example 1: Preparation of Compound E7

[0409] 1. Preparation of intermediates 3-4

[0410] 1.1 Preparation of Compound 2 [ka]

[0411] At 15°C, compound 1 (300g, 2.01mol) was placed in DCM (1.80L), to which benzyl(2,5-dioxopyrrolidine-1-yl) carbonate (600g, 2.40mol) was slowly added, and TEA (203g, 2.01mol, 280mL) was added dropwise. The mixture was then stirred at 25°C for 16 hours. Reaction product 1 (R) was analyzed by TLC (dichloromethane:methanol = 10:1). f The fact that (=0.32) is retained, and that new prominent spots (R f The presence of compound 2 (=0.52) was confirmed. The reaction mixture was washed with saturated sodium bicarbonate solution (1.00 L x 2). The organic phase was washed with brine (1.00 L), dried over anhydrous Na2SO4, and vacuum concentrated. Compound 2 (approximately 385 g) was obtained as yellow oil without further purification.

[0412] 1.2 Preparation of Compound 2A [ka]

[0413] At 0-15°C, DMAP (19.8g, 162 mmol) was added all at once to a pyridine (1.75L) solution of compound 4 (350g, 1.62mol, HCl) and Ac2O (994g, 9.74mol, 912mL), and TEA (164g, 1.62mol, 226mL) was added dropwise. The mixture was stirred at 25°C for 16 hours. LC-MS (product: RT=0.687 min) confirmed that the starting reaction products were completely consumed. At 25°C, HCl (1.40L) was added to the mixture and stirred for 30 minutes. The resulting mixture was then filtered, and the filtered cake was washed with HCl (300mL). The filtered cake was ground with water (1.45L) at 25°C for 30 minutes. The mixture was filtered, and the filtered cake was washed with water (175mL x 3). The filtered cake was recovered, and compound 2A (approximately 580g) was obtained as a white solid.

[0414] 1.3 Preparation of Compound 2B [ka]

[0415] Three reactions were carried out in parallel.

[0416] At 10-15°C, TMSOTf (137g, 616mmol, 111mL) was added dropwise over 0.5 hours to a solution of compound 2A (200g, 514mmol) in DCM (800mL). The mixture was then stirred at 25°C for 3 hours. Compound 2A (R) was analyzed by TLC (dichloromethane:methanol = 20:1). f =0.54) has been completely consumed, and a new spot (R f It was confirmed that compound 2B (=0.24) was formed. The three reactions were combined. The mixture was cooled to 0-15°C and slowly poured into NaHCO3 (300g dissolved in 3.00L of water) at 0-5°C. The organic phase was separated and the aqueous phase was extracted with DCM (1.00L x 3). The combined organic phase was dried with Na2SO4, filtered, and vacuum concentrated. Compound 2B (approximately 507g) for use in the next step was obtained as yellow oil without further purification.

[0417] 1.4 Preparation of Compound 3 [ka]

[0418] At 0-10°C, TMSOTf (84.4g, 380mmol, 69.0mL) was added dropwise to a mixture of compound 2B (250g, 759mmol) and compound 2 (151g, 531mmol) in DCM (1.00L). The mixture was stirred at 20°C for 12 hours. Compound 2(R) was analyzed by TLC (dichloromethane:methanol = 20:1). f =0.33) has been completely consumed, and a new spot (R f It was confirmed that compound 3 (=0.03) was formed. The combined reaction mixture was cooled to 0-5°C, then poured into NaHCO3 (100g in 1L of water) and stirred at 5-10°C for 10 minutes to separate the phases. The aqueous phase was extracted with DCM (500mL x 2). The combined organic phase was dried with Na2SO4, filtered, and vacuum concentrated. Compound 3 (approximately 360g) was obtained as yellow oil without further purification.

[0419] 1 1H NMR: (400 MHz, DMSO).

[0420] δ=7.79-7.37 (m, 1H), 7.35-7.26 (m, 5H), 5.21-5.20 (m, 1H), 5.00-4.95 (m, 3H), 4.55-4.53 (m, 1H), 4.03-3.86 (m, 3H), 3.61-3.59 (m, 1H), 3.59-3.57 (m, 1H), 3.48-3.40 (m, 6H), 3.39-3.31 (m, 2H), 3.14-3.13 (m, 2H), 2.09 (s, 3H), 1.99 (s, 3H), 1.88 (s, 3H), 1.76-1.74 (m, 3H)

[0421] 1.5 Preparation of intermediates 3-4 (TFA salts) [ka]

[0422] Three reactions were carried out in parallel.

[0423] Compound 3 (180 g, 293 mmol, 21.8 mL) and TFA (33.5 g, 293 mmol, 21.8 mL) were added to a Pd / C (18.0 g, 16.3 mmol, 10% content) mixture in THF (1.80 L) under an argon atmosphere. The suspension was degassed and purged three times with hydrogen. The mixture was stirred under H2 (50 Psi) at 30°C for 2 hours. LC-MS (product: RT=0.697 min) confirmed that compound 3 had been consumed and that a product peak was detected. The three reactions were combined. The mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to remove the solvent. Without further purification, intermediate 3-4 (TFA salt) (393 g, 660 mmol, yield 74.8%, purity 99.6%, TFA) was obtained as a yellow solid.

[0424] 1 1H NMR: (400 MHz, DMSO-d6)

[0425] δ = 7.92 (d, J = 9.1 Hz, 4H), 5.27-5.17 (m, 1H), 5.03-4.91 (m, 1H), 4.60-4.50 (m, 1H), 4.09-3.97 (m, 4H), 3.85 (s, 2H), 3.65-3.46 (m, 10H), 3.04-2.92 (m, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.94-1.86 (m, 3H), 1.82-1.71 (m, 4H).

[0426] 2. Preparation of intermediates 3-5

[0427] 2.1 Preparation of Compound 5 [ka]

[0428] At 25°C, DIEA (30.3g, 234mmol, 40.8mL, 6.60eq) was added all at once to a 1.00L solution of compound 4B (10.0g, 35.5mmol, 1.00eq) and compounds 3-4 (46.3g, 78.2mmol, 2.20eq, TFA) prepared above in DCM (1.00L). The mixture was stirred at 25°C for 30 minutes. HBTU (30.3g, 234mmol, 40.8mL, 6.60eq) was added to the mixture. The mixture was stirred at 25°C for 16 hours. The completion of the reaction was confirmed by LCMS (product: RT=0.681 min). The mixture was concentrated under vacuum. The mixture was added to 0.50N HCl (200mL x 2) at 20°C, and then extracted with DCM (3 x 500mL). The combined organic phase was washed with saturated NaHCO3 (3 × 800 mL) to pH=8, then washed with brine (3 × 500 mL), dried over Na2SO4, and vacuum concentrated. The residue was purified by column chromatography (SiO2, DCM:MeOH = 50:1~15:1). The residue was vacuum concentrated at 40°C and purified by preparative MPLC (column: 800 g Agela C18; mobile phase: [water-ACN]; 15-45%, 25 min; 45%, 10 min). Compound 5 (approximately 180 g + 75.0 g + 87.0 g + 40.0 g + 38.0 g) was obtained as a yellow solid by vacuum drying.

[0429] 417.0g of compound 3-4 was divided into 9 batches and converted to compound 5.

[0430] 2.2 Preparation of Intermediate 3-3 [ka]

[0431] Under an argon atmosphere, Pd / C (3.00 g, 10% content) was placed in THF (300 mL), to which compound 5 (73.0 g, 61.7 mmol, 1.00 eq) and TFA (7.04 g, 61.7 mmol, 4.57 mL, 1.00 eq) were added. The suspension was degassed and purged three times with hydrogen. The mixture was stirred under H2 (20 Psi) at 20°C for 16 hours. TLC (dichloromethane:methanol = 8:1, R) fThe reaction was confirmed to be complete by the test (=0.0). The mixture was filtered through Celite, and the filtrate was concentrated under pressure to remove the solvent, yielding compound 3-3 (approximately 33.4g + 129g + 75.0g) as a white solid.

[0432] 1 1H NMR: (400 MHz, DMSO)

[0433] δ=8.53 (t, J = 5.2 Hz, 1H), 8.18 (d, J = 2.4 Hz, 3H), 8.03 (t, J = 5.2 Hz, 1H), 7.84 (dd, J = 3.6 Hz, 2H), 5.22 (d, J = 3.2 Hz, 2H), 4.96 (dd, J = 3.2 Hz, 2H), 4.55 (d, J =8.4 Hz, 2H), 4.02 (t, J =8.8 Hz, 6H), 3.77-3.59 (m, 5H), 3.58-3.45 (m, 21H), 3.40-3.20 (m, 4H), 2.18 (t, J = 7.6 Hz, 2H), 2.17 (d, J =8.0 Hz, 6H), 2.10 (s, 6H), 1.99 (s, 6H), 1.90-1.80 (m, 8H), 1.77 (s, 6H).

[0434] 3. Preparation of Compound E7

[0435] 3.1 Preparation of Compound 3 [ka]

[0436] Compound 1 (2.00 g, 1.87 mmol, prepared according to the method of intermediate 3-3 described above) was dissolved in DCM (20.0 mL) at room temperature. DIEA (0.135 mL, 0.814 mmol) and Compound 2 (0.550 g, 0.814 mmol) were sequentially added to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 16 hours. The MS response of the product was detected by liquid chromatography / tandem mass spectrometry (LC-MS / MS). Thin-layer chromatography (dichloromethane / methanol = 5 / 1) confirmed the disappearance of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 5 / 1) to obtain Compound 3 (approximately 780 mg) as a white solid.

[0437] 1 1H NMR (400 MHz, CD3OD)

[0438] δ=7.28-7.42 (m, 5H), 5.30-5.34 (m, 4H), 5.04-5.14 (m, 6H), 4.63-4.67 (m, 4H), 4.36-4.44 (m, 2H), 4.00-4.20 (m, 23H), 3.91-3.95 (m, 4H), 3.69-3.77 (m, 9H), 3.52-3.67 (m, 32H), 3.34-3.43 (m, 9H), 2.29-2.31 (m, 4H), 2.14 (s, 12H), 2.03 (s, 12H), 1.92-1.96 (m, 24H). LCMS: m / z = 1221.6 (M / 2+H) + .

[0439] 3.2 Preparation of Compound 4 [ka]

[0440] Compound 3 (1.10 g, 0.451 mmol) was dissolved in MeOH (10.0 mL) at room temperature, and 10% mass fraction wet Pd / C (0.050 g, 0.451 mmol) was added to this solution. The reaction mixture was purged with hydrogen three times and then stirred at 25°C for 18 hours under a hydrogen atmosphere (14.696 psi). The MS response of the product was detected by LC-MS / MS. Furthermore, TLC (dichloromethane / methanol = 10 / 1, color: phosphomolybdic acid) confirmed that the starting material had been completely consumed and that new spots had been formed. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4 (approximately 840 mg) as a white solid.

[0441] 1 1H NMR (400 MHz, CD3OD)

[0442] δ=5.32-5.34 (m 4H), 5.06-5.10 (m, 4H), 4.63-4.65 (m, 4H), 4.38-4.40 (m, 2H), 3.99-4.20 (m, 20H), 3.90-3.97 (m, 4H), 3.69-3.76 (m, 6H), 3.50-3.68 (m, 36H), 3.35-3.44 (m, 11H), 2.28-2.38 (m, 4H), 2.15 (s, 12H), 2.03 (s, 12H), 1.90-1.94 (m, 24H). LCMS: m / z = 1154.7 (M / 2+H) + .

[0443] 3.3 Preparation of Compound 6 [ka]

[0444] Compound 5 (232 mg, 0.364 mmol) was dissolved in DCM (10.0 mL) at room temperature. HBTU (207 mg, 0.546 mmol), DIEA (0.181 mL, 1.09 mmol), and Compound 4 (840 mg, 0.364 mmol) were added sequentially to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 1 hour. LC-MS / MS detected the disappearance of the starting material. TLC (dichloromethane / methanol = 5 / 1) confirmed the burning of the starting material and the formation of new spots. The reaction mixture was concentrated under reduced pressure. The resulting crude product was purified by column chromatography (dichloromethane / methanol = 8 / 1-5 / 1) to obtain Compound 6 (approximately 620 mg) as a white solid.

[0445] 1 1H NMR (400 MHz, CD3OD)

[0446] δ=7.41-7.43 (m, 2H), 7.23-7.34 (m, 7H), 6.83-6.90 (m, 4H), 5.31-5.35 (m, 4H), 5.01-5.12 (m, 4H), 4.63-4.65 (m, 4H), 4.41-4.45 (m, 2H), 4.31-4.33 (m, 1H), 3.99-4.22 (m, 22H), 3.87-3.97 (m, 6H), 3.58-3.81 (m, 45H), 3.34-3.43 (m, 10H), 2.19-2.40 (m, 10H), 2.14 (s, 12H), 2.02 (s, 12H), 1.92-1.96 (mz, 24H), 1.48-1.63 (m, 4H), 1.28-1.38 (m, 8H). LCMS: m / z = 1460.0 (M / 2+H) + .

[0447] 4. Preparation of compound E7 [ka]

[0448] Compound 6 (300 mg, 0.103 mmol) was dissolved in DCM (10.0 mL) at room temperature. DIEA (0.102 mL, 0.618 mmol), compound 7 (10.3 mg, 0.103 mmol), and DMAP (12.6 mg, 0.103 mmol) were sequentially added to this solution, and the mixture was purged with nitrogen three times. The reaction mixture was stirred at 25°C for 2 hours. Disappearance of the starting materials was detected by LC-MS / MS. The reaction mixture was concentrated under reduced pressure. The resulting crude product was separated by preparative MPLC (prep-HPLC, column: Waters Xbridge BEH C18 100*30 mm*10 μm; mobile phase: water-ACN; B%: 17%-57%, 5 min) to obtain compound E7 (53.0 mg, yield 17.08%, purity 78.94%) as a white solid.

[0449] 1 1H NMR (400 MHz, CD3OD) δ=7.41-7.45 (m, 2H), 7.17-7.34 (m, 7H), 6.85-6.89 (m, 4H), 5.32-5.36 (m, 4H), 5.03-5.13 (m, 4H), 4.63-4.67 (m, 4H), 4.38-4.47 (m, 2H), 4.32-4.34 (m, 1H), 4.01-4.26 (m, 22H), 3.88-4.00 (m, 6H), 3.77-3.81 (m, 7H), 3.49-3.76 (m, 45H), 3.33-3.47 (m, 10H), 2.56-2.62 (m, 2H), 2.45-2.55 (m, 3H), 2.21-2.38 (m, 7H), 2.14 (s, 12H), 2.05-2.11 (m, 2H), 2.02 (s, 12H), 1.92-1.96 (m, 24H), 1.47-1.68 (m, 4H), 1.28-1.34 (m, 8H)

[0450] MS: m / z = 3022.36 (M+H) + .

[0451] Example 2 Preparation of siRNA

[0452] The siRNAs of this disclosure were prepared using the solid-phase phosphoramidite method, which is well known in this field. Specific methods can be found, for example, in PCT applications WO2016081444 and WO2019105419, but are briefly described below.

[0453] 1. Preparation of ligand-unbound siRNAs 1.1 Synthesis of sense strands (SS strands)

[0454] A solid-phase phosphoramidite synthesis method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the nucleotide sequence order of the sense strand. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for oligonucleotides on a 5 μmol scale are as follows.

[0455] Commercially available phosphoramidites modified with 2'-F and 2'-O-methyl were used. Nucleoside monomers were supplied as a 0.05 mol / L acetonitrile solution. The reaction conditions were the same for each step: deprotection was performed three times at 25°C using a 3% trichloroacetic acid-dichloromethane solution; coupling was performed twice using a 0.25 mol / L ETT-acetonitrile solution as an activator; capping was performed twice using 10% anhydrous acetobiliary and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v); oxidation was performed twice using 0.05 mol / L iodine tetrahydrofuran / pyridine / water (70:20:10, v / v / v); and thiolation was performed twice using 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1:1, v / v).

[0456] 1.2 Synthesis of antisense chains (AS chains) A solid-phase phosphoramidite synthesis method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the nucleotide sequence order of the antisense strand. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for the 5 μmol scale oligonucleotides used as the antisense strand were the same as those for the sense strand.

[0457] 1.3 Purification and annealing of oligonucleotides 1.3.1 Ammonolithesis

[0458] The synthesized solid phase support (sense chain or antisense chain) was added to a 5 mL centrifuge tube, and a 3% diethylamine / ammonia solution (v / v) was added. The reaction was carried out in a constant temperature water bath at 35°C for 16 hours (or at 55°C for 8 hours), and then filtered. The solid phase support was washed three times with 1 mL of ethanol / water each time. The filtrate was concentrated by centrifugation, and the crude product was purified.

[0459] 1.3.2 Purification

[0460] Methods for purification and desalting are well known to those skilled in the art. For example, elution and purification can be performed using a strong anion-packed column and a sodium chloride-sodium hydroxide system, and the product can be recovered and pooled. Desalting can be performed using a gel-packed purification column, with pure water as the elution system.

[0461] 1.3.3 Annealing

[0462] According to Table 6, sense chains (SS chains) and antisense chains (AS chains) were mixed in a molar ratio (SS chain / AS chain = 1 / 1.05), heated in a water bath to 70-95°C for 3-5 minutes, allowed to cool naturally to room temperature, and the system was freeze-dried to obtain the product.

[0463] The following siRNAs in Table 6 were ultimately obtained: DR006925, DR006166, DR006177, DR006255, DR006257, DR006298, DR006421, DR006444, DR006445, DR006446, DR006447, DR006459, DR006560, DR006566, DR006618, DR006621, DR006652, DR006653, DR006657, and DR006658. Of these, DR006925 is a prior art siRNA and was used as a positive control in this disclosure.

[0464] 2. Preparation of siRNA using a ligand-bound sense strand

[0465] 2.1 Ligand binding to CPG carrier

[0466] Binding of compound E7 to the CPG support

[0467] Compound E7 (53 mg, 0.018 mmol) and HBTU (13.3 mg, 0.035 mmol) were mixed and shaken to dissolve in acetonitrile (5 mL). Next, DIEA (9.0 mg, 0.07 mmol) and DMAP (2.1 mg, 0.018 mmol) were added and shaken until dissolved. Blank support resin (550 mg, CPG pore size 1000 Å) was weighed and added to the reaction mixture, and the mixture was left in a shaker at 20°C overnight. A sample was taken and monitored, and thin-layer chromatography (TLC) (developing solvent: DCM / methanol = 4 / 1; color development: phosphomolybdic acid) was performed to confirm that the reaction was complete. The reaction mixture was filtered through a sand-core funnel. The filtration cake was washed with anhydrous acetonitrile (20 mL x 5) and collected. 530 mg of an off-white solid was obtained by suction filtration under reduced pressure using an oil pump for 6 hours.

[0468] The concentrated product (530 mg) was transferred to a 50 mL round-bottom flask. CapC (DMAP / acetonitrile), CapB (N-methylimidazole / pyridine / acetonitrile), and CapA (acetic anhydride / acetonitrile) were added sequentially. The mixture was then left in a shaker at room temperature overnight. After filtration, the filtration cake was washed with acetonitrile (20 mL x 4) and collected. It was then filtered under reduced pressure using an oil pump for 8 hours to obtain 200 mg of an off-white solid, which was used for solid-phase synthesis.

[0469] 2.2 Synthesis of Sense Strands (SS Strands)

[0470] The solid-phase phosphoramidite method was employed, and the E7 solid support prepared above was used as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the nucleotide sequence order of the sense strand. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for oligonucleotides on a 5 μmol scale are as follows.

[0471] Nucleoside monomers were supplied as a 0.05 mol / L acetonitrile solution. The reaction conditions were the same for each step: temperature 25°C; deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution; coupling was performed twice using a 0.25 mol / L ETT-acetonitrile solution as an activator; capping was performed twice using 10% anhydrous acetobiliary and pyridine / N-methylimidazole / acetonitrile (10:14:76, v / v / v); oxidation was performed twice using 0.05 mol / L iodine tetrahydrofuran / pyridine / water (70:20:10, v / v / v); and thiolation was performed twice using 0.2 mol / L PADS acetonitrile / 3-methylpyridine (1:1, v / v).

[0472] 2.3 Synthesis of antisense chains (AS chains)

[0473] A solid-phase phosphoramidite synthesis method was employed, using a blank CPG solid support as the starting cycle. Nucleoside monomers were attached one by one from 3' to 5' according to the nucleotide sequence order of the antisense strand. Each nucleoside monomer attachment involved four steps: deprotection, coupling, capping, and oxidation or thiolation. The synthesis conditions for the 5 μmol scale oligonucleotides used as the antisense strand were the same as those for the sense strand.

[0474] 2.4 Purification and annealing of oligonucleotides

[0475] 2.4.1 Ammonolithesis

[0476] The synthesized solid phase support (sense chain or antisense chain) was added to a 5 mL centrifuge tube, and 3% diethylamine / ammonia (v / v) was added. The reaction was carried out in a constant temperature water bath at 35°C for 16 hours (or at 55°C for 8 hours), and then filtered. The solid phase support was washed three times with 1 mL of ethanol / water each time. The filtrate was concentrated by centrifugation, and the crude product was purified.

[0477] 2.4.2 Purification

[0478] Methods for purification and desalting are well known to those skilled in the art. For example, the product can be eluted and purified using a strong anion-packed column and a sodium chloride-sodium hydroxide system, recovered and pooled, and desalted using a gel-packed purification column, with pure water as the elution system.

[0479] 2.4.3 Annealing

[0480] According to Table 6, sense chains (SS chains) and antisense chains (AS chains) were mixed in a molar ratio (SS chain / AS chain = 1 / 1.05). The mixture was heated in a water bath to 70-95°C, held for 3-5 minutes, and then allowed to cool naturally to room temperature. The product was obtained by freeze-drying the system.

[0481] The following siRNAs were ultimately obtained, as shown in Table 6: DR007102, DR007112-DR007130, DR009198, DR009427, DR009428, and DR009197. Of these, DR007102 is a prior art siRNA and was used as a positive control in this disclosure.

[0482] Example 3: Activity screening in human primary hepatocytes (PHH).

[0483] Cell transfection

[0484] 1.4 mL of rat tail collagen solution (Sigma, C3867) was added to 40.6 mL of DNase / RNase-free distilled water and thoroughly mixed. The mixture was added to 96-well culture plates at a rate of 40 μL / well and coated overnight at 4°C. The coating medium was removed the following day.

[0485] On the second day, prior to use, the coated cell plates were rinsed with DPBS and then aspirated. PHH (Shanghai Xuanyi Biotechnology, Cat#: QYLF-HPMC) was recovered at 37°C and transferred to recovery medium, then centrifuged, resuspended, and counted. PHH was added to 96-well plates at a rate of 90 μL / well (2 × 10⁶). 4 Seeds were sown at a rate of one seed per well. The culture medium was completely replaced after 4 hours, and transfection was performed after 18 hours.

[0486] On the third day, the 20 μM siRNA stock solution was diluted with Opti-MEM. 198 μL of Opti-MEM was added to 2 μL of the 20 μM siRNA stock solution and thoroughly mixed by pipetting to obtain the first concentration point. Serial dilutions were then performed as determined experimentally.

[0487] On day 3, 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently mixed by pipetting, and allowed to stand at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound were gently mixed by pipetting (avoiding the formation of air bubbles) and allowed to stand at room temperature for 10 minutes. The mixture was transferred to a 96-well plate at a rate of 10 μL / well. After incubation in a 5% CO2 incubator at 37°C for 24 hours, RNA was extracted.

[0488] RNA extraction

[0489] Cellular RNA was extracted using a nucleic acid extraction device (Hangzhou Allsheng, Auto-pure96) according to the protocol of a high-throughput cell RNA extraction kit (FireGen, FG0412).

[0490] RNA reverse transcription

[0491] PrimeScript TM The denaturation reaction was prepared according to the II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 1 μL of Oligo dT Primer, 1 μL of dNTP Mixture, and 12.5 μL of template RNA. The denaturation reaction was carried out by incubation at 65°C for 5 minutes in a conventional PCR instrument. The mixture was rapidly cooled on ice for 2 minutes.

[0492] PrimeScript TM Reverse transcription reaction mixtures were prepared according to the Prime Script II 1st Strand cDNA Synthesis Kit (Takara, 6210B). Each well contained 4 μL of 5× Prime Script II Buffer, 0.5 μL of RNase Inhibitor, and 1 μL of PrimeScript II RTase.

[0493] The denaturation reaction mixture (14.5 μL) was gently mixed with the reverse transcription reaction mixture. Reverse transcription was performed by incubation at 42°C for 45 minutes using a conventional PCR instrument, and then the enzyme was inactivated by incubation at 95°C for 5 minutes. Subsequently, the reverse transcript (cDNA) was cooled at 4°C.

[0494] After reverse transcription, 30 μL of DNase / RNase-free distilled water was added to each well's cDNA sample.

[0495] Fluorescence Quantitative PCR

[0496] TaqMan TM A fluorescence quantitative PCR reaction was performed in a 20 μL system (ABI, QuantStudio3) following the instructions for Fast Advanced Master Mix (ABI, 4444965). The reaction program was as follows: (50°C, 2 min) × 1 cycle; (95°C, 20 sec) × 1 cycle; (95°C, 1 sec; 60°C, 24 sec) × 40 cycles.

[0497] Primer information is shown in Table 7. [Table 7]

[0498] Data Statistics

[0499] 2 -△△Ct The value was calculated and converted to a percentage to obtain the residue suppression rate.

[0500] △△Ct=[(Ct 標的遺伝子,実験群 -Ct 参照遺伝子,実験群 )-(Ct 標的遺伝子,コントロール群 -Ct 参照遺伝子,コントロール群 )]

[0501] The target gene was hMASP2, and the reference gene was hACTB.

[0502] The final concentration of siRNA was set to 10 nM, and siRNA activity was screened in primary human hepatocytes. The screening results are shown in Table 8. [Table 8]

[0503] Example 4 Activity screening in primary monkey hepatocytes (PCH)

[0504] Cell transfection

[0505] 100 μL of coating medium was added to each well of a 96-well cell culture plate, incubated at 37°C for 30 minutes, and the coating medium was aspirated after completion.

[0506] Primary monkey hepatocytes (PCH, CCH-100CY-PQ, purchased from MILECELL BIOTECHNOLOGY) were thawed at 37°C. These cells were resuspended in thawing medium, centrifuged, counted, and then loaded into 96-well plates at 90 μL / well (2 × 10⁶). 4 Seeds were sown in individual wells.

[0507] Compound Dilution: A 20 μM compound stock solution was diluted with Opti-MEM. Specifically, 198 μL of Opti-MEM was added to 2 μL of the compound stock solution, and the mixture was thoroughly mixed by pipetting to obtain the first concentration point.

[0508] Transfection procedure: 0.9 μL of RNAiMAX (Thermo, 13778150) was diluted with 14.1 μL of Opti-MEM, gently pipetted to mix thoroughly, and allowed to stand at room temperature for 5 minutes. Then, 15 μL of the prepared RNAi-MAX mixture and 15 μL of the diluted compound were combined and gently pipetted (to avoid creating air bubbles) to mix thoroughly, and allowed to stand at room temperature for 10 minutes. Next, this mixture was added to a 96-well plate at a rate of 10 μL / well. After incubation in a 5% CO2 incubator at 37°C for 4 hours, the culture medium was changed, and RNA was extracted the following day.

[0509] RNA extraction and PCR were performed in the same manner as in Example 3, using the primers shown below. [Table 9]

[0510] The target gene was mkMASP2, and the reference gene was mkGAPDH.

[0511] The starting concentration of the compounds was set at 10 nM, and the activity of 34 siRNA compounds was screened at five concentration points (10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM) obtained by 10-fold dilution. The experimental results are shown in Table 10. In the table, the % values ​​represent the residual inhibition rate, and the calculation method is the same as in Example 3. [Table 10]

[0512] Example 5: Activity screening experiment in primary hepatocytes (PMH) of transgenic mice

[0513] Free eating

[0514] Primary hepatocytes derived from transgenic mice (purchased from Biocytogen Pharmaceuticals (Beijing)) were isolated and counted, and 100 μL / well (1 × 10⁶) were added to a 96-well plate. 4 Seeds were sown in individual wells.

[0515] Ad libitum feeding: 10 μL of diluted compound (final concentration) was added to 90 μL of Opti-MEM, thoroughly mixed, and then added to the corresponding wells. The mixture was incubated in a 5% CO2 incubator at 37°C for 24 hours.

[0516] RNA extraction and PCR were performed in the same manner as in Example 3, using the primers shown below. [Table 11]

[0517] The target gene was hMASP2, and the reference gene was mGAPDH.

[0518] The compound was started at a concentration of 20 nM, and the IC50 activity at five concentration points (20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM) obtained by 5-fold dilution was screened in primary hepatocytes of transgenic mice under free-feeding conditions. The experimental results are shown in Table 12. In the table, the percentage values ​​represent the residue inhibition rate, and the calculation method is the same as in Example 3. [Table 12]

[0519] Example 6: Activity screening in human primary hepatocytes (PHH).

[0520] Following the method described in Example 3, the activity of the siRNA compound was screened at seven concentration points (10 nM, 2.5 nM, 0.625 nM, 0.156 nM, 0.039 nM, 0.00976 nM, 0.00244 nM) obtained by 4-fold dilution, with the starting concentration of the compound being 10 nM. The experimental results are shown in Table 13. [Table 13]

[0521] Example 7: Pharmacological activity screening in AAV mice

[0522] AAV virus containing a fragment of the human MASP2 gene (Obio Technology (Shanghai), H27393, pAAV-TBG-hMASP2-tWPA) was injected into the tail vein of mice at a viral load of 1 × 10^11 vg. The day of virus injection was defined as day -14 of the modeling cycle. Serum was collected from the mice on D-4 / D0, and MASP2 protein (Abcam, ab278121) was detected by ELISA to establish the baseline for the administration groups, with 4 mice per group. On day 0, siRNA or physiological saline was administered subcutaneously at a dose of 3 mg / kg. Serum was collected at each time point after administration (D7, D14, D21, D28, D42, D49), and MASP2 protein levels were detected. The experimental results are shown in Table 13. [Table 14]

[0523] Example 8: Pharmacological activity screening in Tg mice

[0524] Fifty-five MASP2 transgenic mice (Biocytogen Pharmaceuticals (Beijing)) were grouped into groups of five based on their baseline (D0) MASP2 protein levels, with three mice selected for the DR007102 injection group. On day 0, siRNA or saline was administered subcutaneously at a dose of 3 mg / kg. Serum samples were collected at various time points after administration (D7, D14, D21, D28, D42, D56) to detect MASP2 protein levels. The experimental results are shown in Table 14. [Table 15]

Claims

1. A double-stranded nucleotide (dsRNA) for suppressing the expression of mannan-binding lectin-related serine protease 2 (MASP2) in cells, comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand and the antisense strand are each independently 15 to 30 nucleotides long, and the antisense strand contains the nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs. 22 to 40.

2. The dsRNA according to claim 1, wherein the sense strand comprises a nucleotide sequence of at least 15 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 2 to 20.

3. The dsRNA according to claim 1 or 2, wherein the dsRNA is siRNA.

4. The dsRNA according to any one of claims 1 to 3, wherein the antisense strand is 15 to 27 nucleotides long, preferably 17 to 25 nucleotides long, more preferably 18 to 23 nucleotides long, more preferably 19 to 22 nucleotides long, or most preferably 21 amino acids long.

5. The antisense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, a nucleotide sequence of at least 18 adjacent nucleotides, a nucleotide sequence of at least 19 adjacent nucleotides, or a nucleotide sequence of at least 20 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 22 to 40, and preferably comprises a nucleotide sequence described in any one of SEQ ID NOs: 22 to 40, the dsRNA according to any one of claims 1 to 4.

6. The sense strand comprises a nucleotide sequence of at least 16 adjacent nucleotides, a nucleotide sequence of at least 17 adjacent nucleotides, or a nucleotide sequence of at least 18 adjacent nucleotides in the nucleotide sequence described in any one of SEQ ID NOs: 2 to 20, and preferably comprises a nucleotide sequence described in any one of SEQ ID NOs: 2 to 20, the dsRNA according to any one of claims 1 to 5.

7. The above siRNA is the dsRNA according to any one of the following pairs of sense strand sequence and antisense strand sequence. (1) Sense chain: Sequence ID 2, Antisense chain: Sequence ID 22; (2) Sense chain: Sequence ID 3, Antisense chain: Sequence ID 23; (3) Sense chain: Sequence ID 4, Antisense chain: Sequence ID 24; (4) Sense chain: Sequence ID 5, Antisense chain: Sequence ID 25; (5) Sense strand: Sequence ID 6, Antisense strand: Sequence ID 26; (6) Sense strand: Sequence ID 7, Antisense strand: Sequence ID 27; (7) Sense strand: Sequence ID 8, Antisense strand: Sequence ID 28; (8) Sense chain: Sequence ID 9, Antisense chain: Sequence ID 29; (9) Sense chain: Sequence ID 10, Antisense chain: Sequence ID 30; (10) Sense chain: Sequence ID 11, Antisense chain: Sequence ID 31; (11) Sense chain: Sequence ID 12, Antisense chain: Sequence ID 32; (12) Sense chain: Sequence ID 13, Antisense chain: Sequence ID 33; (13) Sense chain: Sequence ID 14, Antisense chain: Sequence ID 34; (14) Sense chain: Sequence ID 15, Antisense chain: Sequence ID 35; (15) Sense chain: Sequence ID 16, Antisense chain: Sequence ID 36; (16) Sense chain: Sequence ID 17, Antisense chain: Sequence ID 37; (17) Sense chain: Sequence ID 18, Antisense chain: Sequence ID 38; (18) Sense strand: Sequence ID 19, Antisense strand: Sequence ID 39; and (19) Sense chain: Sequence ID 20, Antisense chain: Sequence ID 40

8. The dsRNA according to any one of claims 1 to 7, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, or all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

9. The dsRNA according to claim 8, wherein the sense strand and the antisense strand each independently contain one or more nucleotide modifications selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, and phosphorothioate internucleotide bond modifications.

10. The dsRNA according to claim 8, wherein the sense strand and / or antisense strand comprises an SCP-modified nucleotide.

11. The dsRNA according to any one of claims 8 to 10, wherein the 3' and / or 5' ends of the sense strand and / or antisense strand contain 1 to 5 phosphorothioate nucleotide interbonds, preferably 2 to 3 phosphorothioate nucleotide interbonds.

12. The above antisense strand is 21 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21, and 2'-fluoro modified nucleotides at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counted from the 5' end); and / or (ii) Inter-phosphorothioate nucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21. A dsRNA according to any one of claims 1 to 11, comprising:

13. The above antisense strand is 21 nucleotides long, and (i) SCP modification at position 1 (counted from the 5' end); (ii) 2'-fluoro modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, and 18 (counted from the 5' end); (iii) 2'-O-methyl modifications at positions 3, 5, 7, 9, 11, 13, 15, 17, 19, 20, and 21 (counted from the 5' end); and / or (iv) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21. A dsRNA according to any one of claims 1 to 11, comprising:

14. The above antisense strand is 21 nucleotides long, and (i) 2'-deoxy modifications at positions 2, 5, 7, and 12 (counted from the 5' end); (ii) SCP modification at position 1 (counted from the 5' end); (iii) 2'-fluoro modification at position 14 (counted from the 5' end); (iv) 2'-O-methyl modifications at positions 3, 4, 6, 8, 9, 10, 11, 13, 15, 16, 17, 18, 19, 20, and 21 (counted from the 5' end); and / or (v) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21 A dsRNA according to any one of claims 1 to 11, comprising:

15. The sense strand described above is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, and between nucleotide positions 18 and 19 (counted from the 5' end) A dsRNA according to any one of claims 1 to 11, comprising:

16. The sense strand described above is 19 nucleotides long, and (i) 2'-O-methyl modified nucleotides at positions 1-6 and 10-19, and 2'-fluoro modified nucleotides at positions 7-9 (counted from the 5' end); and / or (ii) Phosphothioate internucleotide bonds (counted from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 17 and 18, and between nucleotide positions 18 and 19 A dsRNA according to any one of claims 1 to 11, comprising:

17. The dsRNA according to any one of claims 1 to 14 and 16, wherein the antisense strand is selected from any one of the modified nucleotide sequences described in SEQ ID NOs. 100 to 122, and / or the sense strand is selected from any one of the modified nucleotide sequences described in SEQ ID NOs. 80 to 98.

18. The dsRNA according to any one of the following pairs of modified sense strand sequences and modified antisense strand sequences, as described above. Sense chain: SEQ ID NO: 80, Antisense chain: SEQ ID NO: 100; Sense chain: SEQ ID NO: 81, Antisense chain: SEQ ID NO: 101; Sense chain: SEQ ID NO: 82, Antisense chain: SEQ ID NO: 102; Sense chain: SEQ ID NO: 83, Antisense chain: SEQ ID NO: 103; Sense chain: Sequence ID 84, Antisense chain: Sequence ID 104; Sense strand: Sequence ID 85, Antisense strand: Sequence ID 105; Sense chain: Sequence ID 86, Antisense chain: Sequence ID 106; Sense chain: Sequence ID 87, Antisense chain: Sequence ID 107; Sense chain: Sequence ID 88, Antisense chain: Sequence ID 108; Sense chain: Sequence ID 89, Antisense chain: Sequence ID 109; Sense chain: SEQ ID NO: 90, Antisense chain: SEQ ID NO: 110; Sense chain: SEQ ID NO: 91, Antisense chain: SEQ ID NO: 111; Sense chain: SEQ ID NO: 92, Antisense chain: SEQ ID NO: 112; Sense chain: SEQ ID NO: 93, Antisense chain: SEQ ID NO: 113; Sense chain: Sequence ID 94, Antisense chain: Sequence ID 114; Sense chain: Sequence ID 95, Antisense chain: Sequence ID 115; Sense chain: SEQ ID NO: 96, Antisense chain: SEQ ID NO: 116; Sense strand: SEQ ID NO: 97, Antisense strand: SEQ ID NO: 117; and Sense chain: SEQ ID NO: 98, Antisense chain: SEQ ID NO: 118

19. The dsRNA according to any one of claims 1 to 15, wherein the dsRNA is further bound to a ligand portion containing N-acetylgalactosamine, and preferably the sense strand is bound to the ligand portion.

20. The above ligand has the following structure: 【Chemistry 1】 (In the formula, 【Chemistry 2】 The dsRNA according to claim 19, wherein GL6 has a point that connects to the sense strand (preferably the 3' end of the sense strand) of the dsRNA via a phosphate group or a phosphorothioate group.

21. The dsRNA according to claim 20, wherein the antisense strand comprises a sequence selected from any one of the modified nucleotide sequences described in SEQ ID NOs. 100 to 122, and / or the sense strand comprises a sequence selected from any one of the modified nucleotide sequences described in SEQ ID NOs. 60 to 78.

22. The dsRNA according to claim 21, wherein the dsRNA includes one of the following pairs of modified sense strand sequences and modified antisense strand sequences. Sense chain: Sequence ID 60, Antisense chain: Sequence ID 100; Sense chain: Sequence ID 61, Antisense chain: Sequence ID 101; Sense chain: Sequence ID 62, Antisense chain: Sequence ID 102; Sense chain: SEQ ID NO: 63, Antisense chain: SEQ ID NO: 103; Sense chain: Sequence ID 64, Antisense chain: Sequence ID 104; Sense chain: Sequence ID 65, Antisense chain: Sequence ID 105; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 106; Sense chain: SEQ ID NO: 67, Antisense chain: SEQ ID NO: 107; Sense chain: Sequence ID 68, Antisense chain: Sequence ID 108; Sense strand: Sequence ID 69, Antisense strand: Sequence ID 109; Sense chain: SEQ ID NO: 70, Antisense chain: SEQ ID NO: 110; Sense chain: SEQ ID NO: 71, Antisense chain: SEQ ID NO: 111; Sense chain: SEQ ID NO: 72, Antisense chain: SEQ ID NO: 112; Sense chain: SEQ ID NO: 73, Antisense chain: SEQ ID NO: 113; Sense chain: SEQ ID NO: 74, Antisense chain: SEQ ID NO: 114; Sense chain: SEQ ID NO: 75, Antisense chain: SEQ ID NO: 115; Sense chain: SEQ ID NO: 76, Antisense chain: SEQ ID NO: 116; Sense chain: SEQ ID NO: 77, Antisense chain: SEQ ID NO: 117; Sense chain: Sequence ID 78, Antisense chain: Sequence ID 118; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 119; Sense chain: Sequence ID 66, Antisense chain: Sequence ID 120; Sense strand: Sequence ID 66, Antisense strand: Sequence ID 121; and Sense chain: SEQ ID NO: 66, Antisense chain: SEQ ID NO: 122

23. A cell containing the dsRNA described in any one of claims 1 to 22.

24. A pharmaceutical composition comprising dsRNA according to any one of claims 1 to 22 or cells according to claim 23, and optionally a pharmaceutically acceptable carrier or excipient.

25. A kit comprising dsRNA according to any one of claims 1 to 22, cells according to claim 23, or a pharmaceutical composition according to claim 24.

26. A method for treating a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 22, a cell according to claim 23, or a pharmaceutical composition according to claim 24.

27. A method for preventing at least one symptom in a subject having a disease or disorder in which a reduction in the expression of mannan-binding lectin-related serine protease 2 (MASP2) is beneficial, the method comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 22, a cell according to claim 23, or a pharmaceutical composition according to claim 24.

28. The method according to claim 26 or 27, wherein the disease or disorder for which a reduction in the expression of the above-mentioned mannan-binding lectin-related serine protease 2 (MASP2) is beneficial is a MASP2-mediated disease or a MASP2-related disease.

29. The method according to claim 28, wherein the above-mentioned MASP2-mediated disease or MASP2-related disease is selected from the group consisting of arthritis, IgA nephropathy, thrombotic microangiopathy, venous embolism, diabetic nephropathy, and membranous nephropathy.

30. A method for reducing the level of mannan-binding lectin-related serine protease 2 (MASP2) in a subject, comprising the step of administering to the subject a dsRNA according to any one of claims 1 to 22, a cell according to claim 23, or a pharmaceutical composition according to claim 24.

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