Use of double-stranded ribonucleic acids and modifications, conjugates thereof

Abstract

The present disclosure relates to the use of double-stranded ribonucleic acids and modifications, conjugates thereof. The double-stranded ribonucleic acids, modifications and / or conjugates provided by the present disclosure for inhibiting the expression of the coagulation factor XII gene can target and inhibit the expression of coagulation factor XII in fibrotic tissue, thereby preventing and / or treating pulmonary fibrosis, lung injury and asthma.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine, and specifically, it relates to the use of a double-stranded ribonucleic acid (BRNA), a BRNA modification, or a BRNA conjugate for inhibiting coagulation factor XII gene expression in the prevention and / or treatment of diseases associated with abnormal expression of the coagulation factor XII gene, such as pulmonary fibrosis, lung injury, and asthma. Background Technology

[0002] Coagulation factor XII (also known as FXII, F12, or Hagmann factor), a serine protease primarily expressed in the liver and found in the blood, has a dual function in the intrinsic coagulation pathway and the kallikrein system, which plays a role in inflammation, blood pressure control, coagulation, and pain. The active form of coagulation factor XII binds to and cleaves coagulation factor XI and pre-kallikrein in the coagulation cascade, producing the active forms FXI and kallikrein, respectively.

[0003] Factor FXII is a protease involved in the coagulation pathway in the body. It does not participate in physiological hemostasis but is involved in fibrinolysis and the formation of pathological thrombi. Therefore, FXII plays an important role in the propagation stage of pathological clot formation, while it is not needed for normal hemostasis. This paradigm shift of separating thrombosis and hemostasis has led to the anticoagulation concept of using drugs with minimal bleeding side effects (i.e., the development of FXIIa and FXIa inhibitors) as next-generation anticoagulants.

[0004] Double-stranded RNA (dSRNA), its modified forms, and conjugates that inhibit coagulation factor XII gene expression have been shown to be useful in treating diseases such as atherosclerosis, Alzheimer's disease, and deep vein thrombosis. However, no reports have been found regarding the effects of these DSRNAs, their modified forms, and conjugates on other conditions. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] This disclosure aims to provide a series of double-stranded ribonucleic acid (DRNA), DRNA modifications, and DRNA conjugates for inhibiting coagulation factor XII gene expression, for use in the prevention and / or treatment of pulmonary fibrosis, lung injury, and asthma, for use in the preparation of products for the prevention and / or treatment of pulmonary fibrosis, lung injury, and asthma, and for methods of preventing and / or treating pulmonary fibrosis, lung injury, and asthma.

[0007] Solution for solving the problem

[0008] In a first aspect, this disclosure provides the use of double-stranded ribonucleic acid, double-stranded ribonucleic acid modifications, and / or double-stranded ribonucleic acid conjugates in at least one of the following:

[0009] (1) Used for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene;

[0010] (2) To prepare drugs for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene;

[0011] Among them, the diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury and asthma;

[0012] The double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and / or double-stranded ribonucleic acid conjugate are double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and / or double-stranded ribonucleic acid conjugates used to inhibit the expression of coagulation factor XII gene.

[0013] In some embodiments, the double-stranded ribonucleic acid, double-stranded ribonucleic acid modification, or double-stranded ribonucleic acid conjugate used to inhibit coagulation factor XII gene expression is the double-stranded ribonucleic acid, double-stranded ribonucleic acid modification, or double-stranded ribonucleic acid conjugate disclosed in CN118308353A, the contents of which are incorporated herein by reference in their entirety.

[0014] In some embodiments, the double-stranded RNA includes a sense strand and an antisense strand, wherein the combination of the sense strand and the antisense strand is a combination of the sense strand and the antisense strand selected from any of siRNAs 1 to 21 shown in Table 1; preferably, the sense strand and the antisense strand are selected from siRNA 6.

[0015] In some embodiments, each nucleotide in the sense strand is independently a modified or unmodified nucleotide, and / or each nucleotide in the antisense strand is independently a modified or unmodified nucleotide.

[0016] In some embodiments, any two nucleotides linked together in the sense strand are connected by a phosphodiester bond or a thiophosphate diester bond, and / or, any two nucleotides linked together in the antisense strand are connected by a phosphodiester bond or a thiophosphate diester bond.

[0017] In some embodiments, the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate derivative group along the 5'-to-3' direction, and / or, the 5'-terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate derivative group along the 5'-to-3' direction.

[0018] In some implementations, the double-stranded ribonucleic acid is siRNA.

[0019] In some embodiments, the double-stranded ribonucleic acid modifier is a modification of the double-stranded ribonucleic acid described above, which includes at least one of the following chemical modifications:

[0020] (1) Modification of at least one nucleotide in the positive strand,

[0021] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0022] (3) Modification of at least one nucleotide in the antisense strand,

[0023] (4) Modification of phosphodiester bonds at at least one position in the antisense chain.

[0024] In some embodiments, the modification of the nucleotide is selected from 2'-fluoro modification, 2'-alkoxy modification, 2'-substituted alkoxy modification, 2'-alkyl modification, 2'-substituted alkyl modification, 2'-deoxy modification, nucleotide derivative modification, or any combination of two or more thereof.

[0025] In some embodiments, the modification of the nucleotide is selected from 2'-F modification, 2'-O-CH3 modification, 2'-O-CH2-CH2-O-CH3 modification, 2'-O-CH2-CH=CH2 modification, 2'-CH2-CH2-CH=CH2 modification, 2'-deoxy modification, nucleotide derivative modification, or any combination of two or more thereof.

[0026] In some embodiments, the nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA, or GNA.

[0027] In some embodiments, the ribonucleotides at positions 7, 9, 10, and 11 of the positive strand along the 5' end to the 3' end are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides.

[0028] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 5, 7, 8, and 9 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides.

[0029] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 3, 7, 8, and 9 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides.

[0030] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 3, 7, 9, and 11 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides.

[0031] In some embodiments, the positive chain contains phosphothioester bonds located at the following positions along the 5' end to the 3' end:

[0032] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0033] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0034] Between the first and second nucleotides starting at the 3' end of the positive strand;

[0035] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0036] or,

[0037] The positive chain contains phosphothiophosphate diester bonds located at the following positions:

[0038] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0039] Between the second and third nucleotides starting at the 5' end of the positive strand.

[0040] In some embodiments, along the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0041] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0042] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 of the antisense strand is a ribonucleotide modified by the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0043] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 of the antisense strand is a ribonucleotide modified by the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0044] Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 7, 10, and 14 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0045] In some embodiments, the antisense chain comprises a phosphothioester bond located at the following positions:

[0046] Between the first and second nucleotides starting at the 5' end of the antisense strand;

[0047] Between the second and third nucleotides starting at the 5' end of the antisense strand;

[0048] Between the first and second nucleotides starting at the 3' end of the antisense strand;

[0049] Between the second and third nucleotides starting at the 3' end of the antisense strand.

[0050] In some embodiments, the positive strand of the double-stranded ribonucleic acid modification has a structure as shown in any one of (a1)-(a5):

[0051] (a1)5'-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

[0052] (a2)5'-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N 10 fN 11 f-mN 12 -mN13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',

[0053] (a3)5'-mN1-(s)-mN2-(s)-mN3-mN4-N5f-mN6-N7f-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',

[0054] (a4)5'-mN1-(s)-mN2-(s)-N3f-mN4-mN5-mN6-N7f-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3',

[0055] (a5)5'-mN1-(s)-mN2-(s)-N3f-mN4-mN5-mN6-N7f-mN8-N9f-mN 10 -N 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3';

[0056] Among them, N1-N 19 Ribonucleotides selected independently of each other from bases A, U, C, or G.

[0057] The capital letter T indicates a deoxyribonucleotide with the base thymine.

[0058] The lowercase letter 'm' indicates that the ribonucleotide adjacent to the right of 'm' is a ribonucleotide modified with 2'-O-CH3.

[0059] The lowercase letter 'f' indicates that the ribonucleotide adjacent to the left of 'f' is a 2'-F modified ribonucleotide.

[0060] -(s)- indicates that two adjacent nucleotides are linked by a phosphothioester bond.

[0061] In some embodiments, the antisense strand of the double-stranded ribonucleic acid modification has a structure as shown in any one of (b1)-(b9):

[0062] (b1)5'-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

[0063] (b2)5'-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

[0064] (b3)5'-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

[0065] (b4)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’,

[0066] (b5)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’,

[0067] (b6)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’,

[0068] (b7)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19-(s)-T-(s)-T-3',

[0069] (b8)5'-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3',

[0070] (b9)5'-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-mN6-N7f-mN8-mN9-N 10 f-mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3';

[0071] Among them, N1-N 19 Ribonucleotides selected independently of each other from bases A, U, C, or G.

[0072] The capital letter T indicates a deoxyribonucleotide with the base thymine.

[0073] The lowercase letter 'm' indicates that the ribonucleotide adjacent to the right of 'm' is a ribonucleotide modified with 2'-O-CH3.

[0074] The lowercase letter 'f' indicates that the ribonucleotide adjacent to the left of 'f' is a 2'-F modified ribonucleotide.

[0075] P1 indicates that the nucleotide adjacent to the right of this letter is a 5'-phosphate nucleotide.

[0076] EVP indicates that the nucleotide adjacent to the right of this letter is a 5'-trans-vinylphosphonate nucleotide.

[0077] -(s)- indicates that two adjacent nucleotides are linked by a phosphothioester bond.

[0078] (GNA) indicates that the ribonucleotide adjacent to it on the right is a ribonucleotide modified with GNA.

[0079] In some implementations, the double-stranded ribonucleic acid modifier is an siRNA modifier.

[0080] In some implementations, the combination of the sense strand and the antisense strand is a combination of the sense strand and antisense strand selected from any of siRNAs 22 to 33 as shown in Table 2.

[0081] In some embodiments, the double-stranded ribonucleic acid conjugate includes double-stranded ribonucleic acid or double-stranded ribonucleic acid modifiers as described above; and a conjugation group attached to the double-stranded ribonucleic acid or the double-stranded ribonucleic acid modifier.

[0082] In some embodiments, the conjugating group has the following structure:

[0083]

[0084] In some embodiments, the conjugation group is attached to the 3' end of the positive chain.

[0085] In some embodiments, the conjugating group is conjugated to the 3' end of the positive chain via a phosphodiester bond;

[0086] Preferably, the sense strand and antisense strand of the double-stranded ribonucleic acid conjugate are complementary to form the double-stranded region of the double-stranded ribonucleic acid conjugate, and the 3' end of the sense strand forms a blunt end, while the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region;

[0087] or,

[0088] The sense and antisense strands of the double-stranded ribonucleic acid conjugate are complementary to form the double-stranded region of the double-stranded ribonucleic acid conjugate, and the 3' end of the sense strand is flattened, as is the 3' end of the antisense strand.

[0089] In some embodiments, the double-stranded ribonucleic acid conjugate has the following structure:

[0090]

[0091] The double helix structure is a double-stranded ribonucleic acid or a double-stranded ribonucleic acid modified product.

[0092] In some embodiments, the double-stranded ribonucleic acid conjugate is an siRNA conjugate.

[0093] In some embodiments, the double-stranded ribonucleic acid conjugate is formed by linking any of the siRNAs shown in Table 1 to the conjugate group via a phosphodiester bond or a thiophosphate diester bond; or, the double-stranded ribonucleic acid conjugate is formed by linking any of the siRNA modifiers shown in Table 2 to the conjugate group.

[0094] Preferably, in the double-stranded ribonucleic acid conjugate, the combination of the sense strand and the antisense strand is a combination of the sense strand and antisense strand selected from any one of siRNA 34 to siRNA 42 shown in Table 3.

[0095] In a second aspect, this disclosure provides a method for preventing and / or treating diseases associated with abnormal expression of coagulation factor XII gene, comprising administering a therapeutically effective amount of double-stranded ribonucleic acid, a double-stranded ribonucleic acid modifier, and / or a double-stranded ribonucleic acid conjugate to a subject in need.

[0096] Among them, the diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury and asthma;

[0097] The double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and double-stranded ribonucleic acid conjugate are double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and double-stranded ribonucleic acid conjugate used to inhibit the expression of coagulation factor XII gene.

[0098] Thirdly, this disclosure provides the use of N-ER-FY009151M9L96 in the preparation of a medicament for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene;

[0099] The diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury, and asthma.

[0100] In some embodiments, the double-stranded ribonucleic acid, double-stranded ribonucleic acid modification, or double-stranded ribonucleic acid conjugate is the double-stranded ribonucleic acid, double-stranded ribonucleic acid modification, or double-stranded ribonucleic acid conjugate disclosed in CN118308353A for inhibiting the expression of coagulation factor XII gene, and the content of CN118308353A is incorporated herein by reference in its entirety.

[0101] In some implementations, the subject is a mammal, preferably a human.

[0102] The effects of the invention

[0103] In some embodiments, the double-stranded ribonucleic acid (siRNA) of this disclosure targets and degrades the mRNA of the FXII gene transcription product, exerting RNA interference to inhibit the protein expression of the FXII gene, thereby achieving prevention and / or treatment of pulmonary fibrosis, lung injury, and asthma. Taking pulmonary fibrosis as an example, studies have found that plasma coagulation factor XII levels are not elevated in patients with pulmonary fibrosis, but coagulation factor XII levels are elevated in the lung tissue of fibrotic pulmonary fibrosis. This may be because coagulation factor XII, which is normally confined to circulation, leaks from damaged blood vessels into the fibrotic lung interstitium, inducing fibroblast migration in a concentration-dependent manner, and inducing IL-6 protein expression in fibrotic tissue through PAR-1 and NF-κB, thereby driving the progression of chronic inflammation and fibrosis. This disclosure has experimentally demonstrated that by targeting coagulation factor XII and inhibiting its expression in fibrotic tissue with the double-stranded ribonucleic acid of this disclosure, fibrosis can be improved, thereby preventing and / or treating pulmonary fibrosis, lung injury, and asthma.

[0104] In some embodiments, this disclosure modifies double-stranded ribonucleic acid to obtain double-stranded ribonucleic acid modified products. These modified products have high stability and are suitable for in vivo treatment of pulmonary fibrosis, lung injury, and asthma.

[0105] Furthermore, the double-stranded ribonucleic acid modification is an siRNA modification, which has high stability and good inhibitory activity.

[0106] In some embodiments, this disclosure involves attaching conjugation groups to double-stranded ribonucleic acid (BRNA) or BRNA modifiers to obtain BRNA or BRNA conjugates, which can be used for highly efficient targeted delivery to tissues and cells, reducing the impact of BRNA or BRNA modifiers on non-targeted normal tissues and cells, and improving their safety in the clinical treatment of pulmonary fibrosis, lung injury, and asthma.

[0107] Furthermore, the double-stranded ribonucleic acid conjugate is an siRNA conjugate, which, while maintaining the inhibitory activity and stability of siRNA, also has organ or tissue targeting properties. This can reduce the impact on other tissues or organs and reduce the amount of siRNA molecules used, thereby achieving the goals of reducing toxicity and lowering costs.

[0108] Furthermore, the conjugation group in this disclosure is a group with the structure shown in Formula I (GalNAc). GalNAc can be used for targeted delivery into liver cells and tissues to efficiently inhibit the expression of the FXII gene in the liver. Attached Figure Description

[0109] Figure 1a A demo plot of HE staining area / total area (%) is shown in Example 3 on day 28.

[0110] Figure 1b The graph shows the HE staining area / total area (%) on day 28 in Example 3.

[0111] Figure 2a A demo image showing the percentage of masson staining area to total area on day 28 in Example 3 is displayed.

[0112] Figure 2b The graph shows the percentage of masson staining area to total area on day 28 in Example 3.

[0113] Figure 3a The DEMO plot shows the α-SMA positive area / total area (%) on day 28 in Example 3.

[0114] Figure 3b The graph shows the percentage of α-SMA positive area to total area on day 28 in Example 3.

[0115] Figure 4 The image shows the Ashcroft Score disease score statistics on day 28 in Example 3.

[0116] Figure 5a A demo plot of FXII positive area / total area (%) is shown on day 28 in Example 3.

[0117] Figure 5b The graph shows the percentage of FXII positive area to total area on day 28 in Example 3.

[0118] Figure 6 The graph shows the Penh (standard ventilation index) statistics on day 14 in Example 4.

[0119] Figure 7 The graph shows the number of lymphocytes in the bronchoalveolar lavage fluid on day 17 in Example 4.

[0120] Figure 8a A demo plot of HE staining area / total area (%) is shown on day 17 in Example 4.

[0121] Figure 8b The diagram shows the airway wall thickness statistics on day 17 in Example 4. Detailed Implementation

[0122] definition

[0123] Unless otherwise stated, the terms used in this disclosure have the following meanings.

[0124] In the claims and / or specification of this disclosure, the words “a”, “an”, or “the” may mean “one”, but may also mean “one or more”, “at least one”, and “one or more”.

[0125] As used in the claims and specification, the words “comprising,” “having,” “including,” or “containing” mean included or open-ended and do not exclude additional, uncited elements or method steps.

[0126] Throughout this application, the term "about" means that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define this disclosure are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned test methods or apparatus. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by the term "about." Here, "about" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0127] As used in the context of this disclosure, the term "FXII" refers to a well-known gene and polypeptide. FXII genes and FXII mRNA sequences are readily available, for example, from sources such as GenBank, UniProt, and the Online Mendelian Inheritance Database (OMIM).

[0128] The term "FXII gene" can refer to the wild-type FXII gene or a mutant FXII gene with sequence variations. Many sequence variations in the FXII gene have been identified and can be found in, for example, NCBIdbSNP and UniProt (see, for example, ncbi.nlm.nih.gov / snp).

[0129] The terms "peptide" and "protein" are used interchangeably to refer to a string of at least two amino acid residues linked together by covalent bonds (e.g., peptide bonds), and can be recombinant, natural, or synthetic peptides. Peptides can be linear or branched, can contain modified amino acids, and can be separated by non-amino acid segments. The term also includes amino acid polymers that have been modified (e.g., through disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).

[0130] In some implementations, the target gene is the FXII gene. In some implementations, the target portion of the sequence will be at least long enough to act as a substrate for iRNA-guided cleavage at or near a nucleotide sequence portion of the mRNA molecule formed during transcription of the FXII gene.

[0131] In this art, “G,” “C,” “A,” “T,” and “U” typically represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively. However, it is also generally known in the art that each of “G,” “C,” “A,” “T,” and “U” typically also represents a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of “G,” “C,” “A,” “T,” and “U” include all of the above-mentioned possible cases. However, it should be understood that the terms “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide (as further detailed below) or an alternative substitution. “Ribonucleotide” and “nucleotide” are used interchangeably herein. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide having such a substitution). For example, without limitation, nucleotides including inosine as their base can be base-paired with nucleotides including adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequence of the dsRNA characterized in this disclosure by nucleotides containing, for example, inosine. In another example, adenine and cytosine anywhere in an oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU swing base pairing with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods characterized in this disclosure.

[0132] The terms “iRNA,” “RNAi reagent,” “iRNA agent,” and “RNA interference agent” as used in the context of this disclosure are used interchangeably and refer to terms defined herein that contain siRNA and mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, for example, the expression of target genes in cells (such as the cells of a subject, such as a mammalian subject).

[0133] As used in the context of this disclosure, the terms "double-stranded ribonucleic acid," "double-stranded RNA (dsRNA) molecule," and "dsRNA" are used interchangeably. The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands, described as having "sense" and "antisense" orientation relative to a target gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0134] Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “double-stranded ribonucleic acid” can include chemically modified ribonucleotides, phosphate backbones, etc. These modifications can include all types of modifications disclosed herein or known in the art.

[0135] As used in the context of this disclosure, the term "isonucleotide" refers to a compound formed by altering the position of a base on the ribose ring, for example, a compound formed by attaching a base to the 2' or 3' position of the ribose ring instead of the 1' position.

[0136] In some embodiments, the double-stranded RNA disclosed herein is siRNA, which interacts with the mRNA sequence transcribed from the target gene to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short 19–23 base pairs of interfering RNA with a characteristic dibase 3' overhang (Bernstein et al., (2001) Nature 409:363). These siRNAs are then incorporated into the RNA-inducible silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0137] As used in the context of this disclosure, the term "protruding nucleotide" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. "Blunt-ended" or "flat-tipped" means that there are no unpaired nucleotides at that end of the double-stranded ribonucleic acid, i.e., no nucleotide protrusions. A "flat-tipped" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its entire length, i.e., without nucleotide protrusions at either end of the molecule.

[0138] The term "antisense strand" refers to a strand of double-stranded RNA that is substantially complementary to a target sequence (e.g., derived from human FXII mRNA). Mismatches at the terminal regions are most tolerable when the complementary region is not perfectly complementary to the target sequence, and if mismatches do occur, they are typically within one or more terminal regions, such as 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0139] The term "sense chain" refers to a double-stranded RNA strand containing a region that is substantially complementary to the antisense chain region.

[0140] The term “inhibition” can be used interchangeably with “reduction,” “silence,” “downregulation,” “suppression,” and other similar terms, and includes any level of inhibition.

[0141] The term "suppressing FXII gene expression" includes suppressing the expression of any FXII gene (e.g., the mouse FXII gene, the rat FXII gene, the monkey FXII gene, or the human FXII gene) and variants (e.g., naturally occurring variants) or mutants of the FXII gene. Therefore, the FXII gene can be a wild-type FXII gene, a mutant FXII gene, or a transgenic FXII gene in the case of genetically manipulated cells, cell groups, or organisms.

[0142] "Inhibition of FXII gene expression" includes inhibition of the FXII gene at any level, such as at least partial inhibition of FXII gene expression, such as inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0143] The term "independently" means that at least two groups (or ring systems) in a structure with the same or similar value ranges can have the same or different meanings under specific circumstances. For example, if substituent X and substituent Y are independently hydrogen, hydroxyl, alkyl, or aryl, then when substituent X is hydrogen, substituent Y can be hydrogen, hydroxyl, alkyl, or aryl; similarly, when substituent Y is hydrogen, substituent X can be hydrogen, hydroxyl, alkyl, or aryl.

[0144] The term "alkyl" includes straight-chain, branched, or cyclic saturated alkyl groups. For example, alkyl groups include, but are not limited to, methyl, ethyl, propyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclohexyl, and similar groups. For example, "C 1-6 The "C" in "alkyl" 1-6 "" refers to a group consisting of 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight chain, branched chain or cyclic form.

[0145] The term "alkoxy" herein refers to an alkyl group that is attached to the remainder of a molecule by an oxygen atom (-O-alkyl), wherein the alkyl group is as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, etc.

[0146] The term "treatment" refers to the process of exposing a subject to (e.g., administering medication) double-stranded RNA, double-stranded RNA modifications, double-stranded RNA conjugates, or pharmaceutical compositions after the onset of a disease, thereby reducing the symptoms of the disease compared to when the subject is not exposed. It does not imply the complete suppression of the disease's symptoms. Having a disease means that the body exhibits symptoms of a disease.

[0147] The term "prevention" means that, prior to the onset of a disease, by exposing (e.g., administering medication) a subject to the double-stranded RNA, double-stranded RNA modifier, double-stranded RNA conjugate, or pharmaceutical composition disclosed herein, the symptoms of the disease are reduced compared to when the subject is not exposed, and does not imply the necessity of completely suppressing the disease.

[0148] The term "effective amount" refers to the quantity or dose of the double-stranded ribonucleic acid, double-stranded ribonucleic acid modification, double-stranded ribonucleic acid conjugate, or pharmaceutical composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0149] In this disclosure, the application method can be varied or modified in any applicable manner to meet the needs of the properties of the drug, the convenience of patients and medical personnel, and other relevant factors.

[0150] As used in the context of this disclosure, the terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0151] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0152] Uses of double-stranded ribonucleic acid (BRNA), BRNA modifiers, and / or BRNA conjugates

[0153] The first aspect of this disclosure provides the use of double-stranded ribonucleic acid, double-stranded ribonucleic acid modifications, and / or double-stranded ribonucleic acid conjugates in at least one of the following:

[0154] (1) Used for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene;

[0155] (2) To prepare drugs for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene;

[0156] Among them, the diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury and asthma;

[0157] The double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and / or double-stranded ribonucleic acid conjugate are double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and / or double-stranded ribonucleic acid conjugates used to inhibit the expression of coagulation factor XII gene.

[0158] Double-stranded RNA

[0159] In some implementations, double-stranded RNA is used as a substrate for the endonuclease Dicer, and is cleaved into small fragments of dsRNA, i.e., siRNA. In some implementations, the double-stranded RNA is siRNA. The siRNA assembles to form an RNA-induced silencing complex (RISC), which cleaves the target mRNA, thereby inhibiting the expression of the FXII gene.

[0160] Based on the target sequence derived from human FXII mRNA (NM_000505.4), siRNAs that bind to the target mRNA were designed.

[0161] In some specific implementations, the double-stranded ribonucleic acid is selected from any of the siRNAs shown in Table 1. The siRNA provided in this disclosure has high specificity in binding to the target mRNA (FXII mRNA), has good target mRNA silencing activity, and can significantly inhibit FXII gene expression, for the prevention and / or treatment of pulmonary fibrosis, lung injury, and asthma.

[0162] In some embodiments, this disclosure provides an siRNA composition comprising any one or more of the siRNAs shown in Table 1.

[0163] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0164] In some embodiments, any two nucleotides linked in the sense strand are connected by a phosphodiester bond or a phosphothiodiester bond. In some embodiments, any two nucleotides linked in the antisense strand are connected by a phosphodiester bond or a phosphothiodiester bond.

[0165] In some embodiments, the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group. In some embodiments, the 5' terminal nucleotide of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

[0166] When the 5' terminal nucleotide of the antisense strand is attached to a 5' phosphate group or a 5' phosphate derivative, the following structure is formed:

[0167]

[0168] Formula (1) shows the structure formed by linking a 5' nucleotide to a 5' phosphate group, Formula (2) shows the structure formed by linking a 5' nucleotide to an EVP, and Formula (3) shows the structure formed by linking a 5' nucleotide to a 5' methylene phosphate group. In Formula (1), Formula (2), or Formula (3), Base represents a base, such as A, U, G, C, or T, and R' is a hydroxyl group or is substituted by various groups known to those skilled in the art, such as 2'-fluoro(2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0169] In some embodiments, when the 5' terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group, the structure of the 5' terminal nucleotide is as shown in Formula X:

[0170]

[0171] In formula X, Base represents a base, such as A, U, G, C, or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, or 2'-deoxynucleotide.

[0172] Double-stranded RNA modified products

[0173] In some implementations, the double-stranded RNA modifier is a siRNA modifier. The siRNA modifier can improve the stability of the siRNA while maintaining high FXII mRNA repressive activity.

[0174] In some embodiments, the double-stranded ribonucleic acid (SSRNA) modifier comprises at least one nucleotide modification. The nucleotide modification is selected from at least one of ribose group modification and base modification. In some embodiments, "nucleotide modification" refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group with another group, or a nucleotide in which the base is a modified base. The nucleotide modification does not result in a significant weakening or loss of the siRNA's ability to suppress gene expression. For example, modified nucleotides disclosed in JKWatts, G.F. Deleavey, and MJDamha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected. Nucleotide modification can improve the stability of siRNA and maintain its high repressive efficiency against the FXII gene.

[0175] For example, the modified nucleotide has the following structure:

[0176] Wherein, Base represents a base, such as A, U, G, C or T, and the hydroxyl group at the 2' position of the ribosome is replaced by R. These hydroxyl groups at the 2' position of the ribosome can be replaced by various groups known to those skilled in the art, such as 2'-fluoro (2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0177] In some implementations, the 2'-alkoxy modified nucleotide is a 2'-methoxy (2'-OMe, 2'-O-CH3) modified nucleotide, etc.

[0178] In some embodiments, the 2'-substituted alkoxy modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3), a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0179] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, etc.

[0180] In some embodiments, the modification of the nucleotide is a modification of the bases. Base modifications can be of various types known to those skilled in the art. For example, base modifications include, but are not limited to, m... 6 A、Ψ、m 1 A、m 5 A, ms 2 i 6 A、i 6 A、m 3 C, m 5 C、ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U、mcm 5 U、ncm 5 U、ncm 5 Um, D, mcm 5 s 2 U, Inosine(I), hm 5 C, s 4 U、s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.

[0181] In some embodiments, a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide derivative can be an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide. A BNA refers to a restricted or inaccessible nucleotide. A BNA can contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated into the 2',4' position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.

[0182] LNA is shown in Equation (4), ENA is shown in Equation (5), and cET is shown in Equation (6):

[0183]

[0184] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (7), and GNA is shown in formula (8).

[0185]

[0186] In formulas (7) and (8) above, R is selected from H, OH or alkoxy (O-alkyl).

[0187] In equations (4), (5), (6), (7) and (8) above, Base represents a base, such as A, U, G, C or T.

[0188] In some implementations, nucleotide derivative modification refers to the replacement of nucleotides in nucleic acids with nucleotide derivatives. Exemplary examples include isonucleotides, LNA, ENA, cET, UNA, or GNA.

[0189] In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, also referred to as isonucleotide modification in the context of this disclosure. In some embodiments, isonucleotide modification includes incorporating an isonucleotide at one or more sites on the sense and / or antisense strands of the siRNA to be modified, in place of the native nucleoside for coupling at the corresponding position.

[0190] In some embodiments, the isonucleoside modification is D-isonucleoside modification. In other embodiments, the isonucleoside modification is L-isonucleoside modification. In still other embodiments, the isonucleoside modification is a combination of D-isonucleoside and L-isonucleoside modification.

[0191] In some embodiments, the double-stranded ribonucleic acid (siRNA) modifier includes modification of a phosphodiester bond at at least one position. In some embodiments, the modification of the phosphodiester bond refers to the substitution of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a phosphothiodiester bond. The phosphothiodiester bond can stabilize the double-stranded structure of siRNA and maintain the specificity of base pairing. An exemplary phosphothiodiester bond structure is shown below:

[0192]

[0193] In some embodiments, the double-stranded ribonucleic acid modifier comprises at least one of the following chemical modifications:

[0194] (1) Modification of at least one nucleotide in the positive strand,

[0195] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0196] (3) Modification of at least one nucleotide in the antisense strand,

[0197] (4) Modification of phosphodiester bonds at at least one position in the antisense chain.

[0198] Furthermore, the double-stranded RNA modifier is an siRNA modifier containing at least one of the chemical modifications in (1)-(4).

[0199] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 7, 9, 10, and 11 of the sense strand are 2'-fluoromodified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxymodified ribonucleotides.

[0200] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 5, 7, 8, and 9 of the sense strand are 2'-fluoromodified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxymodified ribonucleotides.

[0201] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 3, 7, 8, and 9 of the sense strand are 2'-fluoro-modified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxy-modified ribonucleotides.

[0202] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 3, 7, 9, and 11 of the sense strand are 2'-fluoromodified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxymodified ribonucleotides.

[0203] In some embodiments, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0204] In some implementations, the positive strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5'-to-3' direction: between the first and second nucleotides starting at the 5' end, and between the second and third nucleotides starting at the 5' end.

[0205] In some specific implementations, the siRNA modifier sense strand has a structure as shown in any of (a1)-(a5) above.

[0206] In some implementations, the nucleotide at the 5' end of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group along the 5' end toward the 3' end.

[0207] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0208] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0209] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0210] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

[0211] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 of the antisense strand is 5'-trans-vinylphosphonate nucleotide.

[0212] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 of the antisense strand is 5'-trans-vinylphosphonate nucleotide.

[0213] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 7, 10, and 14 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides, and the ribonucleotide at position 1 of the antisense strand is 5'-trans-vinylphosphonate nucleotide.

[0214] In some embodiments, the antisense strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0215] In some specific implementations, the antisense strand of the siRNA modifier has a structure as shown in any of (b1)-(b9) above.

[0216] In some implementations, double-stranded RNA modifiers include, but are not limited to, siRNA modifiers as shown in Table 2.

[0217] Double-stranded ribonucleic acid conjugates

[0218] In some embodiments, the double-stranded ribonucleic acid conjugate is obtained by linking the double-stranded ribonucleic acid provided in this disclosure to a conjugate group via a phosphodiester bond or a thiophosphate diester bond, or by linking the double-stranded ribonucleic acid modifier provided in this disclosure to a conjugate group.

[0219] In this disclosure, the sense and antisense strands of the double-stranded ribonucleic acid conjugate form a double-stranded region, and a blunt end is formed at the 3' end of the sense strand. In some embodiments, the 3' end of the sense strand of the double-stranded ribonucleic acid conjugate is blunt, and the 3' end of the antisense strand has one or two protruding nucleotides extending out of the double-stranded region. In other embodiments, the 3' end of both the sense and antisense strands of the double-stranded ribonucleic acid conjugate is blunt.

[0220] In some preferred embodiments, the double-stranded ribonucleic acid conjugate is obtained by conjugating a double-stranded ribonucleic acid modifier with a conjugating group. Specifically, the sense and antisense strands of the double-stranded ribonucleic acid modifier are complementary to form the double-stranded region of the modifier, and the 3' end of the sense strand of the modifier is blunt-ended. The conjugating group is then conjugated to the 3' end of the blunt-ended sense strand to form the double-stranded ribonucleic acid conjugate.

[0221] For example, the siRNA conjugate shown as N-ER-FY009151M2L96 is a conjugate obtained by modifying N-ER-FY009151M2 and a conjugating group. Based on the sequence of N-ER-FY009151M2, it is known that the 3' end of the positive strand of this conjugate originally had a protruding nucleotide -sTsT extending into a double-stranded region. Before binding with the conjugating group, the protruding -sTsT nucleotide at the 3' end of the positive strand is removed, resulting in a conjugate containing...

[0222] The blunt-ended sequence 5'-mCsmUsmUmCmUmCCfmGGfUfUfmUmUmCmCmAmCmA-3' (SEQ ID NO:52) serves as the nucleotide sequence for linking the L96 conjugate group (i.e., L96 is linked via a phosphodiester bond after the sequence is synthesized to the blunt end). Therefore, the sequence forming the siRNA conjugate is: [Sense strand]

[0223] 5'-mCsmUsmUmCmUmCCfmGGfUfUfmUmUmUmCmCmAmCmAL96-3' (SEQ ID NO:57), antisense strand 5'-P1mUsGfsmUmGmGAfmAmAmAmAmCmCmGGfmAGfmAmAmGsTsT-3' (SEQ ID NO:44).

[0224] In some alternative embodiments, the positive strand of the double-stranded ribonucleic acid conjugate has the structure shown below:

[0225] (d1)5'-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -L96-3',

[0226] (d2)5'-mN1-(s)-mN2-(s)-mN3-mN4-N5f-mN6-N7f-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17-mN 18 -mN 19 -L96-3',

[0227] (d3)5'-mN1-(s)-mN2-(s)-N3f-mN4-mN5-mN6-N7f-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -L96-3',

[0228] (d4)5'-mN1-(s)-mN2-(s)-N3f-mN4-mN5-mN6-N7f-mN8-N9f-mN 10 -N 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -L96-3';

[0229] Among them, N1-N 19 The ribonucleotides are independently selected from those with bases A, U, C, or G. The uppercase letter T indicates a deoxyribonucleotide with the base thymine. The lowercase letter m indicates that the ribonucleotide adjacent to the right of m is modified with 2'-O-CH3, and the lowercase letter f indicates that the ribonucleotide adjacent to the left of f is modified with 2'-F. -(s)- indicates that the two adjacent nucleotides are linked by a phosphothioester bond. L96 is also the conjugate group GalNAc shown in Formula I.

[0230] In some alternative embodiments, the antisense strand of the double-stranded ribonucleic acid conjugate has a structure as shown in any of (b1)-(b9) above.

[0231] Furthermore, the double-stranded ribonucleic acid conjugate is an siRNA conjugate, wherein the siRNA molecule linked to the conjugation group in the siRNA conjugate can be unmodified siRNA or a modified siRNA. The siRNA molecule modified with the conjugation group maintains high inhibitory activity and stability while also exhibiting good tissue and organ targeting and the ability to promote endocytosis, thus reducing the impact on other tissues or organs and decreasing the amount of siRNA molecule used, thereby achieving the goals of reducing toxicity and lowering costs. Optionally, any siRNA molecule shown in Table 1 or Table 2 can be linked to the conjugation group to obtain a double-stranded ribonucleic acid conjugate.

[0232] The conjugation site of siRNA and the conjugating group can be at the 3' or 5' end of the siRNA's sense strand, at the 5' end of the antisense strand, or within the siRNA's internal sequence. In some embodiments, the conjugation site of siRNA and the conjugating group is at the 3' end of the siRNA's sense strand.

[0233] In some embodiments, the conjugate group may be attached to a phosphate group, a 2'-hydroxyl group, or a base of a nucleotide. In some embodiments, the conjugate group may also be attached to a 3'-hydroxyl group, in which case the nucleotides are linked by a 2',5'-phosphodiester bond. When the conjugate group is attached to the end of the siRNA chain, it is usually attached to a phosphate group of the nucleotide; when the conjugate group is attached to the inner sequence of the siRNA, it is usually attached to a ribose ring or a base. Various connection methods can be found in the reference: Muthiah Manoharanet.al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes. ACS Chemical biology, 2015, 10(5):1181-7.

[0234] In this disclosure, the conjugation group can be a ligand conventionally used in the field of siRNA drug delivery. In some embodiments, the conjugation group can be selected from one or more ligands formed from the following target molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acids, vitamins (e.g., vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as transmembrane peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid; receptor ligands expressed by hepatocytes, such as desialyl glycoprotein, desialyl sugar residues, lipoproteins (e.g., high-density lipoprotein, low-density lipoprotein, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, transferrin, etc.

[0235] In some specific embodiments, the conjugating group has the structure shown in Formula I:

[0236]

[0237] The conjugation group shown in Formula I is GalNAc. GalNAc has liver targeting properties and can deliver siRNA molecules to liver tissue with high specificity, specifically inhibiting the high expression of the FXII gene in the liver.

[0238] In some specific implementations, GalNAc is conjugated to the 3' end of the positive sense strand via a phosphodiester bond, resulting in an siRNA conjugate with the structure shown in Formula II:

[0239]

[0240] The double helix structure is either unmodified siRNA or siRNA modified.

[0241] In some implementations, the double-stranded ribonucleic acid conjugates include, but are not limited to, siRNA conjugates as shown in Table 3.

[0242] Prevention and / or treatment methods

[0243] A second aspect of this disclosure provides a method for preventing and / or treating diseases associated with abnormal expression of coagulation factor XII gene, comprising administering a therapeutically effective amount of double-stranded ribonucleic acid, a double-stranded ribonucleic acid modifier, and / or a double-stranded ribonucleic acid conjugate to a subject in need.

[0244] Among them, the diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury and asthma;

[0245] The double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and double-stranded ribonucleic acid conjugate are double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and double-stranded ribonucleic acid conjugate used to inhibit the expression of coagulation factor XII gene.

[0246] In this disclosure, "subject" includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. Subjects may include transgenic organisms. Most preferably, the subject is a human.

[0247] Table 1 siRNA sequence information

[0248]

[0249]

[0250] Table 2 siRNA Modifiers

[0251]

[0252]

[0253] In the table above, the uppercase letters “G”, “C”, “A”, “T”, and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively; mA, mU, mC, and mG indicate nucleotides modified with 2'-methoxy groups; Af, Gf, Cf, and Uf indicate nucleotides modified with 2'-fluorine groups; the lowercase letter 's' indicates that the two nucleotides adjacent to it are linked by a phosphothioester bond; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphonucleotide; EVP indicates that the nucleotide adjacent to the right of this letter is a 5'-trans-vinylphosphonate nucleotide; and (GNA) indicates that the ribonucleotide adjacent to its right is a ribonucleotide modified with GNA.

[0254] Table 3 siRNA conjugates

[0255]

[0256]

[0257] In the table above, the uppercase letters “G”, “C”, “A”, “T”, and “U” typically represent nucleotides containing guanine, cytosine, adenine, thymine, and uracil as bases, respectively; mA, mU, mC, and mG indicate nucleotides modified with 2'-methoxy groups; Af, Gf, Cf, and Uf indicate nucleotides modified with 2'-fluorine groups; the lowercase letter 's' indicates that the two nucleotides adjacent to it are linked by a phosphothioester bond; P1 indicates that the nucleotide adjacent to the right of P1 is a 5'-phosphonucleotide; EVP indicates that the nucleotide adjacent to the right of EVP is a 5'-trans-vinylphosphonate nucleotide; L96 is the conjugate group GalNAc shown in Formula I.

[0258] In Tables 1, 2, and 3, if the left side of the 5' terminal nucleotide of the positive strand, the modified positive strand, and the modified positive strand with a conjugated group is not marked with P1 or EVP, it means that the 5' terminal nucleotide is not connected to a 5' phosphate group or a 5' phosphate-derived group (i.e., the ribosome of the 5' terminal nucleotide is a 5' hydroxyl group), and its structure is shown in Formula X:

[0259]

[0260] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or hydrogen or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino.

[0261] In Tables 1, 2, and 3, if the left side of the 5' terminal nucleotide of the antisense strand and the modified antisense strand is not marked with P1 or EVP, it means that the 5' terminal nucleotide is not attached to a 5' phosphate group or a 5' phosphate derivative group, and its structure is as shown in Formula X.

[0262] In Tables 1, 2, and 3, the 3' end nucleotides of the sense strand and the modified sense strand, and the 3' end nucleotides of the antisense strand and the modified antisense strand, have a hydroxyl group at the 3' position.

[0263] Example

[0264] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of this disclosure) are given for illustrative purposes only, as various changes and modifications that can be made within the spirit and scope of this disclosure will become apparent to those skilled in the art upon reading this detailed description.

[0265] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0266] The siRNA, siRNA modifiers, and siRNA conjugates disclosed herein are identical to those disclosed in CN118308353A, the contents of which are incorporated herein by reference in their entirety and will not be repeated here.

[0267] According to the descriptions in Examples 3 and 6 of CN118308353A, the experimental results of the above-mentioned siRNA and siRNA modifiers inhibiting the expression of the FXII gene are shown in Table 4 below.

[0268] Table 4. siRNA and siRNA modifications inhibit FXII gene expression (Hep3B cells)

[0269]

[0270] As can be seen from Table 4, the siRNA and siRNA modifiers provided in this disclosure exhibit excellent inhibitory effects on the FXII gene.

[0271] According to Example 7 in CN118308353A, the experimental results of the above-mentioned siRNA conjugate inhibiting FXII gene expression are shown in Table 5 below.

[0272] Table 5. siRNA conjugates inhibit FXII gene expression (PHH cells)

[0273]

[0274]

[0275] As can be seen from Table 5, the siRNA conjugates provided in this disclosure exhibit excellent inhibitory effects on the FXII gene.

[0276] Example 1: siRNA inhibits FXII gene expression

[0277] 1.1 Experimental Materials:

[0278] Hep3B cells, purchased from ATCC, catalog number HB-8064;

[0279] RNAiMAX transfection reagent, purchased from Invitrogen, catalog number 13778-150;

[0280] Opti-medium: serum-reduced culture medium, purchased from Gibco, catalog number 31985-070;

[0281] EMEM culture medium: purchased from ATCC, catalog number 30-2003;

[0282] RNA extraction kit 96 Kit, item number QIAGEN-74106;

[0283] Fastking RT Kit (with gDNase), purchased from TianGen, item number KR116-02;

[0284] Phosphate Buffer Saline (PBS), purchased from Gibco, catalog number 10010-023;

[0285] GAPDH TaqMan probe primers, purchased from Thermo, catalog number Hs99999905_m1;

[0286] FXII TaqMan probe primers, purchased from Thermo, catalog number HS01557542_g1;

[0287] FastStart Universal Probe Master, purchased from Roche, item number 04914058001.

[0288] 1.2 Experimental Methods:

[0289] 1.2.1 Hep3B cells were seeded in fresh EMEM medium in 96-well plates and cultured for 24 hours. The cultured cells were then resuspended in PS-free (penicillin-streptomycin mixture) EMEM medium to a density of 5.55 × 10⁻⁶ cells / well. 4 Spread 90 μL of cell suspension per well into a 96-well plate, resulting in 5000 cells per well.

[0290] 1.2.2 The dry powder of the siRNA modifier and siRNA conjugate (hereinafter referred to as siRNA in the experimental procedure description of this embodiment for ease of description) was centrifuged at low temperature and high speed, and then dissolved in ultrapure distilled water to prepare a 100 μM stock solution.

[0291] 1.2.3 Prepare 20 nM siRNA dilution solution Z and 2 nM siRNA dilution solution W

[0292] (1) Preparation of 1 μM siRNA stock solution Q and 0.1 μM siRNA stock solution X:

[0293] a) Take 2 μL of the 100 μM siRNA stock solution obtained in step 1.2.2 above, add 18 μL of ultrapure distilled water to obtain a siRNA dilution solution with a final concentration of 10 μM.

[0294] b) Take 2 μL of the 10 μM siRNA dilution solution obtained in step a), add 18 μL of ultrapure distilled water to obtain siRNA stock solution Q with a final concentration of 1 μM.

[0295] c) Take 2 μL of the 1 μM siRNA stock solution Q prepared in step b), add 18 μL of ultrapure distilled water to obtain siRNA stock solution X with a final concentration of 0.1 μM.

[0296] (2) Take 2 μL of each of the above-prepared siRNA stock solution Q and siRNA stock solution X, and add 98 μL of Opti-medium to obtain 20 nM siRNA dilution Z and 2 nM siRNA dilution W respectively.

[0297] 1.2.4 Transfection of Hep3B cells

[0298] (1) Take 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and the 20 nM siRNA diluent Z prepared in step 1.2.3 were mixed at a volume ratio of 1:1 to prepare a transfection mixture. After standing for 5 minutes, 10 μL of the transfection mixture was added to a 96-well plate to transfect the Hep3B cells cultured in step 1.2.1 (final volume 100 μL, the concentration of siRNA in this system is 1 nM).

[0299] (2) Take 3 μL of RNAiMAX transfection reagent was added to 97 μL of Opti-medium to obtain... RNAiMAX transfection reagent dilution solution; The RNAiMAX transfection reagent diluent and the 2nM siRNA diluent W prepared in step 1.2.3 were mixed at a 1:1 volume ratio to prepare a transfection mixture. After standing for 5 minutes, 10 μL of the transfection mixture was added to a 96-well plate to transfect the Hep3B cells cultured in step 1.2.1 (final volume 100 μL, the concentration of siRNA in this system is 0.1 nM).

[0300] The cells were cultured for 48 hours after transfection; two replicates were set up for each concentration (1 nM and 0.1 nM).

[0301] 1.2.5 Utilization According to the 96Kit instructions, extract total RNA from Hep3B cells obtained in step 1.2.4.

[0302] 1.2.6 The extracted total RNA was reverse transcribed into cDNA using the Fastking RT Kit (with gDNase), following these steps:

[0303] a) Remove gDNA using gDNAase according to the table below;

[0304] Table 6

[0305] Volume / μL 5×gDNA Buffer 2 Sample (RNA) 8

[0306] 42℃, 2min;

[0307] b) Add the reagents described below to the system obtained in step a) and perform reverse transcription:

[0308] Table 7

[0309] Volume / μL The mixture from the previous step 10μL 10×RT Mix 2μL HiScriptⅢEnzyme Mix 2μL Oligo(dT) 20 VN]] 1μL Random hexamers 1μL <![CDATA[RNase-free ddH2O]]> 4μL

[0310] 50℃, 15min; 85℃, 5s; 4℃, stand.

[0311] c) Store the reverse transcription product obtained in step b) at -20°C for real-time PCR analysis.

[0312] 1.2.7 Perform real-time PCR analysis

[0313] a) Prepare the qPCR reaction mixture as shown in the table below. Throughout the entire process, all reagents should be kept on ice.

[0314] Table 8

[0315] Volume / μL 2×FastStart universal probe master 5 20× target special gene TaqMan probe primers (FXII) 0.5 <![CDATA[cDNA and H2O obtained in Step 2.2.6]]> 4.5

[0316] Table 8-1

[0317]

[0318] b) Perform the qPCR procedure as described below.

[0319] 50℃, 2 minutes; 95℃, 10 minutes;

[0320] 95℃, 15 seconds; 60℃, 1 minute (40 cycles).

[0321] 1.2.8 Results Analysis

[0322] a) Use Quant Studio 7 software with default settings to automatically calculate the Ct value;

[0323] b) Calculate the relative expression level of the gene using the following formula:

[0324] ΔCt=Ct(FXII gene)–Ct(GAPDH)

[0325] ΔCt = ΔCt(sample group) - ΔCt(mock group)

[0326] mRNA expression relative to the Mock group = 2 -ΔΔCt .

[0327] Inhibition rate (%) = (Relative expression level of mRNA in the Mock group - Relative expression level of mRNA in the test sample group) / Relative expression level of mRNA in the Mock group × 100%

[0328] The Mock group stated: compared to the test sample group, the group without added siRNA.

[0329] 1.3 Experimental Results

[0330] The concentrations of 0.1 nM and 1 nM were selected for testing, and the results are shown in Table 9 below.

[0331] Table 9. siRNA inhibits FXII gene expression.

[0332] siRNA ID 1 nM (%) 0.1 nM (%) N-ER-FY009151M11 75.82 57.91 N-ER-FY009151M44 86.43 52.45 N-ER-FY009151M45 80.13 63.47 N-ER-FY009151M11L96 76.44 59.14 N-ER-FY009151M44L96 82.99 73.02 N-ER-FY009151M45L96 71.58 66.71

[0333] As can be seen from Table 9, the siRNA modifiers and siRNA conjugates provided in this disclosure exhibit excellent inhibitory effects on the FXII gene.

[0334] Example 2: Inhibitory effect of siRNA conjugate on human FXII (hFXII) gene expression in humanized mice

[0335] Six- to eight-week-old C57BL / 6-hFXII mice (provided by Shanghai Southern Model Biotechnology Co., Ltd.) were placed in a breeding facility and acclimatized for seven days. Then, mice were subcutaneously administered N-ER-FY009151M7L96, N-ER-FY009151M9L96, and N-ER-FY009151M11L96 at a single dose of 3 mg / kg (n=6 per group). Serum hFXII protein expression levels were measured on days 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 post-administration. The inhibition rate of hFXII protein expression by the siRNA conjugate was calculated using the formula: inhibition rate % = (1 - average hFXII protein expression after administration / average hFXII protein expression before administration) × 100%, as shown in Table 10.

[0336] Table 10 Inhibition rate of siRNA conjugates on hFXII protein

[0337]

[0338] As can be seen from Table 10, the siRNA conjugate provided by the present invention exhibits excellent inhibitory effects on hFXII protein.

[0339] Example 3: Testing a mouse model of pulmonary fibrosis

[0340] Six- to eight-week-old C57BL / 6-hFXII mice (provided by Shanghai Southern Model Biotechnology Co., Ltd.) were introduced into the breeding facility. After 7 days of acclimatization feeding, bleomycin was administered intratracheally at a dose of 0.66 mg / kg (equivalent to 1 U / kg) in 50 μL on day 1 to induce the bleomycin model. Administration began 5 days after model establishment, with the administration pattern shown in the table below:

[0341] Table 11

[0342] sc: subcutaneous injection; po: oral administration; Qd: once daily.

[0343] After the experimental endpoint, mouse lung tissue was collected, fixed, and stained (including HE staining, Masson's staining, α-SMA staining, and FXII immunohistochemical staining) for histopathological analysis. The results were also statistically analyzed using the Ashcroft score. The results are as follows: Figure 1a , Figure 1b , Figure 2a , Figure 2b , Figure 3a , Figure 3b , Figure 4 , Figure 5a and Figure 5b As shown.

[0344] Conclusion: The endpoint experiment results showed that, by HE staining, the staining area of ​​drug N-ER-FY009151M9L96 was significantly smaller than that of the blank group. Figure 1a , Figure 1b The drug N-ER-FY009151M9L96, compared to the positive control Nintedanib (purchased from Shanghai Mairui Biochemical Technology Co., Ltd.), significantly reduced the inflammatory response. Masson staining showed that the staining area of ​​N-ER-FY009151M9L96 was significantly smaller than that of the blank control group. Figure 2a , Figure 2b (Compared to the positive control Nintedanib), both significantly reduced fibrosis levels. (Through...) Figure 3a and Figure 3b It can be seen that, for the α-SMA positive area, the staining area of ​​the drug N-ER-FY009151M9L96 was significantly smaller compared with the blank group, and comparable to the positive control Nintedanib, which can significantly reduce fibroblasts. This was confirmed by the Ashcroft Score disease score (…). Figure 4 As can be seen, the drug N-ER-FY009151M9L96 significantly reduced the level compared to the blank control group, while the positive control Nintedanib did not show a significant reduction. F12 immunohistochemical staining showed that N-ER-FY009151M9L96 significantly regulated the FXII protein level in the lungs compared to the blank control group, and was superior to the positive control Nintedanib. Figure 5a , Figure 5b ).

[0345] Example 4: House dust mite mouse model test

[0346] Six- to eight-week-old C57BL / 6-hFXII mice (provided by Shanghai Southern Model Biotechnology Co., Ltd.) were placed in the breeding facility and acclimatized for 7 days. Afterward, they were divided into groups of five mice each, based on body weight. Each mouse received an intratracheal injection of 25 μL of HDM solution (prepared as sterile saline solution with a concentration of 1 mg / mL house dust mite) on days 0, 7, and 14. Seven days after the final HDM sensitization, mice were challenged with an intratracheal injection of 25 μL of HDM solution per mouse. Drug administration began after challenge (Day 1), following the dosing schedule shown in the table below:

[0347] Table 12

[0348] Group Drug Name Administration method Dosage Dosage time end G1 blank sc, once on day 1 NA Day 1 Day 17 G2 N-ER-FY009151M9L96 sc, once on day 1 10mpk Day 1 Day 17 G3 Dexamethasone IP, QD 2.5mpk Day 7 to Day 17 Day 17

[0349] sc: subcutaneous injection; ip: intraperitoneal injection; Qd: once daily.

[0350] On day 14 of the experiment, airway hyperresponsiveness was measured in mice using whole-body plethysmography, and the value was expressed as Penh (standard ventilation index). The results showed that, 2 hours after airway injection of HDM, compared with the model group, both the N-ER-FY009151M9L96 and Dexamethasone (purchased from MCE, catalog number HY-14648) administration groups significantly reduced Penh values, with N-ER-FY009151M9L96 showing a more significant decrease than Dexamethasone. Figure 6 ).

[0351] On Day 17, bronchoalveolar lavage fluid (BALF) was collected for cytological analysis. BALF cell counting results showed that intratracheal injection of HDM increased the number of inflammatory cells in mouse BALF. In hF12 mice, compared with the model group, both the N-ER-FY009151M9L96 and Dexamethasone administration groups significantly reduced the number of lymphocytes. Figure 7 ).

[0352] On Day 17, mouse lung tissue was collected and H&E staining was performed. Results showed that after intratracheal injection of HDM, significant inflammatory responses were observed in the left lung tissue of mice, including infiltration of inflammatory cells and thickening of the airway mucosa. In hF12 mice, compared with the model group, both N-ER-FY009151M9L96 and Dexamethasone could alleviate the infiltration of inflammatory cells in lung tissue to some extent. Figure 8a This can reduce airway wall thickness to some extent. Figure 8b ).

[0353] The embodiments described above are merely examples for clearly illustrating the present disclosure and are not intended to limit the implementation of the present disclosure. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. Use of double-stranded ribonucleic acid, double-stranded ribonucleic acid modifications, and / or double-stranded ribonucleic acid conjugates in at least one of the following: (1) Used for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene; (2) To prepare drugs for the prevention and / or treatment of diseases associated with abnormal expression of coagulation factor XII gene; in, The diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury, and asthma; The double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and / or double-stranded ribonucleic acid conjugate are double-stranded ribonucleic acid, double-stranded ribonucleic acid modifier, and / or double-stranded ribonucleic acid conjugates used to inhibit the expression of coagulation factor XII gene.

2. The use according to claim 1, wherein, The double-stranded ribonucleic acid includes a sense strand and an antisense strand, wherein the sense strand and the antisense strand are selected from the following combinations: 1) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:1, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:12; 2) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:2, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:13; 3) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:3, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:14; 4) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:4, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:15; 5) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:5, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:16; 6) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:6, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:17; 7) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:7, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:18; 8) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:8, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:19; 9) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:9, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:20; 10) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:10, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:21; 11) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:11, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:22; 12) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:23, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:24; 13) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:25, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:26; 14) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:27, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:28; 15) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:29, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:30; 16) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:31, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:32; 17) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:33, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:34; 18) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:35, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:36; 19) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:37, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:38; 20) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:39, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:40; 21) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:41, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:42; Preferably, the double-stranded ribonucleic acid is siRNA.

3. The use according to claim 1 or 2, wherein, The double-stranded ribonucleic acid modifier is the double-stranded ribonucleic acid modifier according to claim 2; Preferably, the double-stranded ribonucleic acid modifier comprises at least one of the following chemical modifications: (1) Modification of at least one nucleotide in the positive strand, (2) Modification of the phosphodiester bond at at least one position in the positive chain. (3) Modification of at least one nucleotide in the antisense strand, (4) Modification of the phosphodiester bond at at least one position in the antisense chain; More preferably, the modification of the nucleotide is selected from 2'-fluoro modification, 2'-alkoxy modification, 2'-substituted alkoxy modification, 2'-alkyl modification, 2'-substituted alkyl modification, 2'-deoxy modification, nucleotide derivative modification, or any combination of two or more thereof; More preferably, the modification of the nucleotide is selected from 2'-F modification, 2'-O-CH3 modification, 2'-O-CH2-CH2-O-CH3 modification, 2'-O-CH2-CH=CH2 modification, 2'-CH2-CH2-CH=CH2 modification, 2'-deoxy modification, nucleotide derivative modification, or any combination of two or more thereof; More preferably, the nucleotide derivative in the nucleotide derivative modification is selected from isonucleotides, LNA, ENA, cET, UNA, or GNA.

4. The use according to claim 3, wherein, Along the direction from the 5' end to the 3' end, the ribonucleotides at positions 7, 9, 10, and 11 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 5, 7, 8, and 9 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 3, 7, 8, and 9 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 3, 7, 9, and 11 of the positive strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the positive strand are 2'-O-CH3 modified ribonucleotides.

5. The use according to claim 3 or 4, wherein, Along the 5' end to the 3' end direction, the positive chain contains phosphothioester bonds located at the positions shown below: Between the first and second nucleotides starting at the 5' end of the positive strand; Between the second and third nucleotides starting at the 5' end of the positive strand; Between the first and second nucleotides starting at the 3' end of the positive strand; Between the second and third nucleotides starting at the 3' end of the positive strand; or, The positive chain contains phosphothiophosphate diester bonds located at the following positions: Between the first and second nucleotides starting at the 5' end of the positive strand; Between the second and third nucleotides starting at the 5' end of the positive strand.

6. The use according to any one of claims 3-5, wherein, Along the 5'-to-3' direction, the 5'-terminal nucleotide of the antisense strand is not linked to a 5' phosphate group or a 5' phosphate-derived group; and / or, Along the 5' end toward the 3' end, the nucleotide at the 5' end of the antisense strand is linked to a 5' phosphate group or a 5' phosphate-derived group.

7. The use according to any one of claims 3-6, wherein, Along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14 and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 of the antisense strand is a ribonucleotide modified by the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 of the antisense strand is a ribonucleotide modified by the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides. Alternatively, along the direction from the 5' end to the 3' end, the ribonucleotides at positions 2, 7, 10, and 14 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-O-CH3 modified ribonucleotides.

8. The use according to any one of claims 3-7, wherein, The antisense chain contains a phosphothiophosphate diester bond located at the following positions: Between the first and second nucleotides starting at the 5' end of the antisense strand; Between the second and third nucleotides starting at the 5' end of the antisense strand; Between the first and second nucleotides starting at the 3' end of the antisense strand; Between the second and third nucleotides starting at the 3' end of the antisense strand.

9. The use according to any one of claims 3-8, wherein the positive strand of the double-stranded ribonucleic acid modification has a structure as shown in any one of (a1)-(a5): (a1)5’-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N 10 f-N 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (a2)5'-mN1-(s)-mN2-(s)-mN3-mN4-mN5-mN6-N7f-mN8-N9f-N 10 fN 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3', (a3)5'-mN1-(s)-mN2-(s)-mN3-mN4-N5f-mN6-N7f-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3', (a4)5’-mN1-(s)-mN2-(s)-N3f-mN4-mN5-mN6-N7f-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3’, (a5)5'-mN1-(s)-mN2-(s)-N3f-mN4-mN5-mN6-N7f-mN8-N9f-mN 10 -N 11 f-mN 12 -mN 13 -mN 14 -mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -3', in, N1-N 19 Ribonucleotides selected independently of each other from bases A, U, C, or G. The capital letter T indicates a deoxyribonucleotide with the base thymine. The lowercase letter 'm' indicates that the ribonucleotide adjacent to the right of 'm' is a ribonucleotide modified with 2'-O-CH3. The lowercase letter 'f' indicates that the ribonucleotide adjacent to the left of 'f' is a 2'-F modified ribonucleotide. -(s)- indicates that two adjacent nucleotides are linked by a phosphothioester bond.

10. The use according to any one of claims 3-9, wherein the antisense strand of the double-stranded ribonucleic acid modification has a structure as shown in any one of (b1)-(b9): (b1)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (b2)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (b3)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-(GNA)N7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (b4)5’-P1mN1-(s)-N2f-(s)-mN3-mN4-mN5-(GNA)N6-N7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (b5)5’-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (b6)5'-mN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3', (b7)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-mN8-mN9-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’, (b8)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-N6f-mN7-N8f-N9f-mN 10 -mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -N 16 f-mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’; (b9)5’-EVPmN1-(s)-N2f-(s)-mN3-mN4-mN5-mN6-N7f-mN8-mN9-N 10 f-mN 11 -mN 12 -mN 13 -N 14 f-mN 15 -mN 16 -mN 17 -mN 18 -mN 19 -(s)-T-(s)-T-3’; in, N1-N 19 Ribonucleotides selected independently of each other from bases A, U, C, or G. The capital letter T indicates a deoxyribonucleotide with the base thymine. The lowercase letter 'm' indicates that the ribonucleotide adjacent to the right of 'm' is a ribonucleotide modified with 2'-O-CH3. The lowercase letter 'f' indicates that the ribonucleotide adjacent to the left of 'f' is a 2'-F modified ribonucleotide. P1 indicates that the nucleotide adjacent to the right of this letter is a 5'-phosphate nucleotide. EVP indicates that the nucleotide adjacent to the right of this letter is a 5'-trans-vinylphosphonate nucleotide. -(s)- indicates that two adjacent nucleotides are linked by a phosphothioester bond. (GNA) indicates that the ribonucleotide adjacent to its right is a ribonucleotide modified with GNA; Preferably, the double-stranded ribonucleic acid modifier is an siRNA modifier.

11. The use according to any one of claims 3-10, wherein, The justice chain and the antisense chain are selected from the following combinations: 1) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:44; 2) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:45; 3) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:46; 4) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:47; 5) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:48; 6) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:49; 7) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:50; 8) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:51, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:50; 9) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:52, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:50; 10) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:43, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:53; 11) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:54, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:55; 12) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:56, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:

55.

12. The use according to any one of claims 1-11, wherein, The double-stranded ribonucleic acid conjugate comprises the double-stranded ribonucleic acid as described in claim 2, or the double-stranded ribonucleic acid modifier as described in any one of claims 3-11; and a conjugation group attached to the double-stranded ribonucleic acid or the double-stranded ribonucleic acid modifier; Preferably, the conjugated group has the structure shown in Formula I: Preferably, the conjugation group is attached to the 3' end of the positive chain; Preferably, the conjugating group is conjugated to the 3' end of the positive chain via a phosphodiester bond; More preferably, the sense strand and antisense strand of the double-stranded ribonucleic acid conjugate are complementary to form the double-stranded region of the double-stranded ribonucleic acid conjugate, and the 3' end of the sense strand is blunt, while the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region; or, the sense strand and antisense strand of the double-stranded ribonucleic acid conjugate are complementary to form the double-stranded region of the double-stranded ribonucleic acid conjugate, and the 3' end of the sense strand is blunt, while the 3' end of the antisense strand is blunt.

13. The use according to claim 12, wherein, The double-stranded ribonucleic acid conjugate has the structure shown in Formula II: The double helix structure is a double-stranded ribonucleic acid or a double-stranded ribonucleic acid modified product; Preferably, the double-stranded ribonucleic acid conjugate is an siRNA conjugate.

14. The use according to claim 12 or 13, wherein, The double-stranded ribonucleic acid conjugate is formed by linking any one of the siRNAs shown in Table 1 to the conjugate group via a phosphodiester bond or a thiophosphate diester bond, or the double-stranded ribonucleic acid conjugate is formed by linking any one of the siRNA modifiers shown in Table 2 to the conjugate group. Preferably, in the double-stranded ribonucleic acid conjugate, the sense strand and the antisense strand are selected from the following combinations: 1) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:44; 2) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:45; 3) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:48; 4) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:49; 5) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:50; 6) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:58, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:50; 7) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:57, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:53; 8) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:59, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:55; 9) The sense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:60, and the antisense strand comprises or is composed of a nucleotide sequence as shown in SEQ ID NO:

55.

15. A method for preventing and / or treating a disease associated with abnormal expression of coagulation factor XII gene, comprising administering to a subject in need a therapeutically effective amount of double-stranded ribonucleic acid, a double-stranded ribonucleic acid modifier, and / or a double-stranded ribonucleic acid conjugate; in, The diseases associated with abnormal expression of coagulation factor XII gene include at least one of pulmonary fibrosis, lung injury, and asthma; The double-stranded ribonucleic acid, double-stranded ribonucleic acid modified product and / or double-stranded ribonucleic acid conjugate are as defined in any one of claims 1-14; Preferably, the subject is a mammal, and more preferably a human.

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