Sirna for inhibiting prekallikrein and use thereof

Abstract

The present application belongs to the technical field of biology and relates to an siRNA for inhibiting prekallikrein and the use thereof. Provided in the present application is an siRNA for inhibiting prekallikrein, wherein the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence shown as any one of SEQ ID NOs: 1-294, and the antisense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence shown as any one of SEQ ID NOs: 295-588. Also provided in the present application is a modified siRNA for inhibiting prekallikrein, the modification including methoxy modification, fluorination modification and / or thiophosphate linkage. Experiments have proved that the siRNA and the modified substance thereof of the present application all have high inhibitory activity on prekallikrein. Therefore, the siRNA and the modified substance thereof of the present application all have great application prospects in the preparation of drugs for treating diseases related to the expression of prekallikrein.

Description

A siRNA for inhibiting prokallikrein and its applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510100812.1, filed on January 21, 2025, entitled “An siRNA for inhibiting prokallikrein and its application thereto”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to an siRNA for inhibiting prokallikrein and its applications, belonging to the field of biotechnology. Background Technology

[0004] Bradykinin (BK) is a major regulator of vascular permeability. Prokaryotic release enzyme (PKK) is a precursor of plasma kallikrein (PK) and is encoded by the KLKB1 gene. PKK is activated by factor XIIa (FXIIa) to convert into plasma kallikrein, which cleaves high molecular weight kininogen to release bradykinin into the blood vessels.

[0005] Studies have shown that excessive bradykinin can enhance vascular leakage, thereby exacerbating inflammation. Furthermore, research indicates that a genetic defect in C1-esterase inhibitors (C1-INH), the main natural inhibitor of bradykinin, can lead to hereditary angioedema (HAE). Patients with this rare condition frequently experience acute attacks of painful edema triggered by unknown factors, and attacks located in the throat can be life-threatening. Simultaneously, numerous studies have confirmed that the expression of prokallikrein and its conversion to plasma kallikrein play a crucial role in the development of vascular and inflammatory diseases such as edema (including angioedema, macular edema, and cerebral edema), thromboembolism (including arterial embolism, venous embolism, and cerebral embolism), infarction (including myocardial infarction), and stroke (including cerebrovascular stroke). Therefore, inhibiting the expression of the KLKB1 gene can suppress excessive bradykinin at the cellular level, thereby preventing and treating diseases caused by excessive bradykinin, especially hereditary angioedema.

[0006] Small interfering RNA (siRNA) is a double-stranded RNA of 20-25 nucleotides in length. Based on the mechanism of RNA interference (RNAi), it can inhibit or block the expression of any gene of interest in a sequence-specific manner, thereby achieving the goal of treating diseases. Developing siRNA that can effectively inhibit prokallikrein expression would be a more effective and specific targeted drug for the prevention and treatment of diseases caused by excessive bradykinin (especially hereditary angioedema). Summary of the Invention

[0007] To address the aforementioned problems, this application provides an siRNA for inhibiting prokallikrein, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 1 to 294; and the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 295 to 588.

[0008] In one embodiment of this application, the nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO. 1 to 294; the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NO. 295 to 588.

[0009] In one embodiment of this application, at least one nucleotide in the sense or antisense strand of the siRNA is a modified nucleotide.

[0010] In one embodiment of this application, all nucleotides in the sense strand and / or antisense strand of the siRNA are modified nucleotides, and these modifications on the nucleotide groups do not cause a significant weakening or loss of the function of the siRNA disclosed herein in inhibiting the expression of the prokallikrein encoding gene (i.e., the KLKB1 gene).

[0011] In one embodiment of this application, the modification includes methoxy modification, fluorination modification, thiophosphate linkage and / or 2′-deoxynucleotide modification.

[0012] In one embodiment of this application, the "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with other groups, or a nucleotide whose bases are modified bases.

[0013] In one embodiment of this application, "fluorinated nucleotide" refers to a nucleotide formed by replacing the hydroxyl group at the 2' position of the ribosome with fluorine, and "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the hydroxyl group at the 2' position of the ribosome with a non-fluorinated group.

[0014] In one embodiment of this application, the "nucleotide analogue" refers to a group that can replace a nucleotide in a nucleic acid, but whose structure is different from that of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide, such as isonucleotide, bridged nucleic acid (BNA), or acyclic nucleotide.

[0015] In one embodiment of this application, the "methoxy-modified nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0016] In one embodiment of this application, the "thiophosphate group linkage" means that at least a portion of the phosphate groups in the phosphate-sugar backbone of at least one single strand of the sense and antisense strands of the siRNA are phosphate groups with modifying groups.

[0017] In one embodiment of this application, the "phosphate ester group with a modifying group" is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphate diester bond of the phosphate ester group with a sulfur atom.

[0018] In one embodiment of this application, the "2'-deoxynucleotide modified nucleotide" includes five types: A(d), C(d), G(d), U(d), and T(d), wherein A(d) represents 2'-deoxyadenosine-3'-phosphate, C(d) represents 2'-deoxycytidine-3'-phosphate, U(d) represents 2'-deoxyuridine-3'-phosphate, G(d) represents 2'-deoxyguanosine-3'-phosphate, and T(d) represents 2'-deoxythymidine-3'-phosphate.

[0019] In one embodiment of this application, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides.

[0020] Alternatively, the fluorinated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides of the antisense strand are fluorinated nucleotides.

[0021] In one embodiment of this application, the methoxy-modified nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 of the sense strand are methoxy-modified nucleotides, and at least the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides.

[0022] Alternatively, the methoxy-modified nucleotide is located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 of the sense strand are methoxy-modified nucleotides, and at least the nucleotides at positions 1, 3, 4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand are replaced with methoxy-modified nucleotides.

[0023] In one embodiment of this application, the nucleotides linked by the thiophosphate group are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

[0024] In one embodiment of this application, the 2'-deoxynucleotide modified nucleotide is located in the antisense strand of the nucleotide sequence, and at least the 5th and 7th nucleotides of the antisense strand are 2'-deoxynucleotide modified nucleotides.

[0025] This application also provides an siRNA conjugate containing the above-described siRNA and a conjugate group conjugated to the siRNA.

[0026] In one embodiment of this application, the conjugating group is N-glucose amino acid (N-Acetylgalactosamine, GalNAc).

[0027] This application also provides a recombinant plasmid that expresses the above-mentioned siRNA.

[0028] In one embodiment of this application, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a bacteriophage vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, hybrid virus vector, baculovirus vector, herpes simplex virus vector, or lentivirus vector; the non-viral vector includes a plasmid vector.

[0029] In one embodiment of this application, the plasmid vector includes pUC plasmid, a pUC plasmid-derived plasmid, pAAV plasmid, a pAAV plasmid-derived plasmid, PGEM plasmid, and / or a PGEM plasmid-derived plasmid.

[0030] In one embodiment of this application, the recombinant plasmid is prepared by: designing shRNA based on siRNA; and ligating the shRNA with a linearized vector to obtain the recombinant plasmid.

[0031] This application also provides a host cell whose genome is integrated with the above-mentioned siRNA; or, the host cell carries the above-mentioned siRNA conjugate; or, the host cell carries the above-mentioned recombinant plasmid.

[0032] In one embodiment of this application, the host cell includes fungi, bacteria, plant cells, and / or animal cells.

[0033] This application also provides the use of the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid or the above-mentioned host cell in the preparation of medicaments for the prevention and / or treatment of diseases related to prokallikrein.

[0034] In one embodiment of this application, the diseases associated with prokallikrein include vascular diseases and / or inflammatory diseases.

[0035] In one embodiment of this application, the diseases associated with prokallikrein include edema, thromboembolism, infarction, and / or stroke.

[0036] In one embodiment of this application, the edema includes angioedema, macular edema, and / or cerebral edema; the thromboembolism includes arterial embolism, venous embolism, and / or cerebral vascular embolism; the infarction includes myocardial infarction; and the stroke includes cerebrovascular stroke.

[0037] In one embodiment of this application, the angioedema includes hereditary angioedema.

[0038] This application also provides a prokaryotic release enzyme inhibitor, wherein the inhibitor comprises the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid and / or the above-mentioned host cell.

[0039] In one embodiment of this application, the inhibitor further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include carriers, diluents, binders, and / or lubricants.

[0040] This application also provides a medicament for the prevention and / or treatment of a disease related to the expression of prokallikrein; the medicament comprises the above-mentioned siRNA, the above-mentioned siRNA conjugate, the above-mentioned recombinant plasmid and / or the above-mentioned host cell.

[0041] In one embodiment of this application, the diseases associated with prokallikrein expression include vascular diseases and / or inflammatory diseases.

[0042] In one embodiment of this application, the diseases associated with prokallikrein expression include edema, thromboembolism, infarction, and / or stroke.

[0043] In one embodiment of this application, the edema includes angioedema, macular edema, and / or cerebral edema; the thromboembolism includes arterial embolism, venous embolism, and / or cerebral vascular embolism; the infarction includes myocardial infarction; and the stroke includes cerebrovascular stroke.

[0044] In one embodiment of this application, the angioedema includes hereditary angioedema.

[0045] In one embodiment of this application, the drug composition further includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include carriers, diluents, binders and / or lubricants.

[0046] This application also provides a method for preventing or treating diseases related to prokallikrein, comprising administering the siRNA, the siRNA conjugate, the recombinant plasmid, the host cell, the prokallikrein inhibitor, or the drug to a subject in need.

[0047] In one embodiment of this application, the dosage of the siRNA, siRNA conjugate, recombinant plasmid, host cell, prokallikrein inhibitor, or drug is 0.1-10 mg / kg.

[0048] In one embodiment of this application, the dosage of the siRNA, siRNA conjugate, recombinant plasmid, host cell, prokallikrein inhibitor, or drug is 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0049] In one embodiment of this application, the diseases associated with prokallikrein expression include vascular diseases and / or inflammatory diseases.

[0050] In one embodiment of this application, the diseases associated with prokallikrein expression include edema, thromboembolism, infarction, and / or stroke.

[0051] In one embodiment of this application, the edema includes angioedema, macular edema, and / or cerebral edema; the thromboembolism includes arterial embolism, venous embolism, and / or cerebral vascular embolism; the infarction includes myocardial infarction; and the stroke includes cerebrovascular stroke.

[0052] In one embodiment of this application, the angioedema includes hereditary angioedema.

[0053] The technical solution of this application has the following advantages:

[0054] This application provides an siRNA for inhibiting prokallikrein, wherein the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 1 to 294; and the antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO. 295 to 588. This application aims to provide an siRNA that targets and cleaves KLKB1 mRNA, thereby selectively and effectively inhibiting the expression of the KLKB1 gene and achieving the purpose of disease treatment. In vitro and in vivo experiments have confirmed that the siRNA and its modifications provided in this application both possess high inhibitory activity against prokallikrein. Therefore, the siRNA and its modifications provided in this application have great application potential in the preparation of drugs for treating diseases related to prokallikrein expression. Attached Figure Description

[0055] Figure 1: Effects of different siRNA conjugates on the expression level of the target gene KLKB1 in hKLKB1 transgenic female mice. Detailed Implementation

[0056] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0057] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0058] In the following examples, uppercase letters C, G, U, and A represent ribonucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are modified with a thiophosphate group; string (d) indicates that the nucleotide adjacent to the left of string (d) is a 2'-deoxynucleotide modified nucleotide. "2'-deoxynucleotide modified nucleotides" include five types: A(d), C(d), G(d), U(d), and T(d). Specifically, A(d) represents 2'-deoxyadenosine-3'-phosphate, C(d) represents 2'-deoxycytidine-3'-phosphate, U(d) represents 2'-deoxyuridine-3'-phosphate, G(d) represents 2'-deoxyguanosine-3'-phosphate, and T(d) represents 2'-deoxythymidine-3'-phosphate.

[0059] Example 1: A siRNA for inhibiting prokallikrein

[0060] This embodiment provides a siRNA for inhibiting prokallikrein, wherein the nucleotide sequence of the siRNA is based on the target mRNA (the GeneBank numbers of the target mRNAs are NM_000892.5, NM_001318394.2, NM_001318396.2, XM_011531930.3, XM_017008181.2, XM_017008182.2, XM_0170081...). The designs were obtained from 83.2, XM_017008184.2, XM_047415661.1, XM_015139651.2, XM_028848987.1, NM_008455.3, NM_012725.2, XM_006253121.4, XM_008771253.3, XM_005556481.3, and XM_045393138.1, and are shown in Tables 1 to 30.

[0061] Table 1. siRNAs and their sequences that inhibit prokallikrein.

[0062] Table 2. siRNAs and their sequences that inhibit prokallikrein.

[0063] Table 3. siRNAs and their sequences that inhibit prokallikrein.

[0064] Table 4. siRNAs and their sequences that inhibit prokaloplasmin release.

[0065] Table 5. siRNAs and their sequences that inhibit prokallikrein.

[0066] Table 6. siRNAs and their sequences that inhibit prokallikrein.

[0067] Table 7. siRNAs and their sequences that inhibit prokallikrein.

[0068] Table 8. siRNAs and their sequences that inhibit prokallikrein.

[0069] Table 9. siRNAs and their sequences that inhibit prokallikrein.

[0070] Table 10. siRNAs and their sequences that inhibit prokallikrein.

[0071] Table 11. siRNAs and their sequences that inhibit prokallikrein.

[0072] Table 12. siRNAs and their sequences that inhibit prokallikrein.

[0073] Table 13. siRNAs and their sequences that inhibit prokallikrein.

[0074] Table 14. siRNAs and their sequences that inhibit prokallikrein.

[0075] Table 15. siRNAs and their sequences that inhibit prokallikrein.

[0076] Table 16. siRNAs and their sequences that inhibit prokallikrein.

[0077] Table 17. siRNAs and their sequences that inhibit prokallikrein.

[0078] Table 18. siRNAs and their sequences that inhibit prokallikrein.

[0079] Table 19. siRNAs and their sequences that inhibit prokallikrein.

[0080] Table 20. siRNAs and their sequences that inhibit prokallikrein.

[0081] Table 21. siRNAs and their sequences that inhibit prokallikrein.

[0082] Table 22. siRNAs and their sequences that inhibit prokallikrein.

[0083] Table 23. siRNAs and their sequences that inhibit prokallikrein.

[0084] Table 24. siRNAs and their sequences that inhibit prokallikrein.

[0085] Table 25. siRNAs and their sequences that inhibit prokallikrein.

[0086] Table 26. siRNAs and their sequences that inhibit prokallikrein.

[0087] Table 27. siRNAs and their sequences that inhibit prokallikrein.

[0088] Table 28. siRNAs and their sequences that inhibit prokallikrein.

[0089] Table 29. siRNAs and their sequences that inhibit prokallikrein.

[0090] Table 30. siRNAs and their sequences that inhibit prokallikrein.

[0091] Example 2: A modified siRNA for inhibiting prokallikrein

[0092] This embodiment provides a modified siRNA for inhibiting prokallikrein. The modified siRNA is based on Example 1, but with the nucleotides at positions 7, 8, and 9 of the positive strand replaced by fluorinated nucleotides, and the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 replaced by methoxylated nucleotides. A sulfuric acid junction is used between the nucleotides at positions 1 and 2, and between positions 2 and 3. Phosphophosphate linkage: The nucleotides at positions 2, 6, 14, and 16 of the antisense strand of the siRNA are replaced with fluorinated nucleotides, and the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 are replaced with methoxylated nucleotides. The nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate linkages, as shown in Tables 31 to 60.

[0093] Alternatively, the modified siRNA is based on Example 1, but with the nucleotides at positions 7, 8, and 9 of the siRNA's sense strand replaced by fluorinated nucleotides, and the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 replaced by methoxylated nucleotides. The nucleotides at positions 1 and 2, and positions 2 and 3 are linked by phosphate thioester groups. The nucleotides at positions 2, 10, and 19 of the siRNA's antisense strand are also modified. Nucleotides at positions 12, 14, 16, 18, and 20 are replaced with fluorinated nucleotides; nucleotides at positions 1, 3, 4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 are replaced with methoxylated nucleotides; nucleotides at positions 5 and 7 are replaced with 2'-deoxynucleotides; and nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 are linked by thiophosphate groups, as shown in Tables 38 and 41.

[0094] Table 31. Modified siRNAs and their sequences that inhibit prokallikrein.

[0095] Table 32. Modified siRNAs and their sequences that inhibit prokallikrein.

[0096] Table 33. Modified siRNAs and their sequences that inhibit prokallikrein.

[0097] Table 34. Modified siRNAs and their sequences that inhibit prokallikrein.

[0098] Table 35. Modified siRNAs and their sequences that inhibit prokallikrein.

[0099] Table 36. Modified siRNAs and their sequences that inhibit prokallikrein.

[0100] Table 37. Modified siRNAs and their sequences that inhibit prokallikrein.

[0101] Table 38. Modified siRNAs and their sequences that inhibit prokallikrein.

[0102] Table 39. Modified siRNAs and their sequences that inhibit prokallikrein.

[0103] Table 40. Modified siRNAs and their sequences that inhibit prokallikrein.

[0104] Table 41. Modified siRNAs and their sequences that inhibit prokallikrein.

[0105] Table 42 Modified siRNAs and their sequences that inhibit prokallikrein.

[0106] Table 43. Modified siRNAs and their sequences that inhibit prokallikrein.

[0107] Table 44. Modified siRNAs and their sequences that inhibit prokallikrein.

[0108] Table 45. Modified siRNAs and their sequences that inhibit prokallikrein.

[0109] Table 46. Modified siRNAs and their sequences that inhibit prokallikrein.

[0110] Table 47. Modified siRNAs and their sequences that inhibit prokallikrein.

[0111] Table 48. Modified siRNAs and their sequences that inhibit prokallikrein.

[0112] Table 49. Modified siRNAs and their sequences that inhibit prokallikrein.

[0113] Table 50. Modified siRNAs and their sequences that inhibit prokallikrein.

[0114] Table 51. Modified siRNAs and their sequences that inhibit prokallikrein.

[0115] Table 52. Modified siRNAs and their sequences that inhibit prokallikrein.

[0116] Table 53. Modified siRNAs and their sequences that inhibit prokallikrein.

[0117] Table 54. Modified siRNAs and their sequences that inhibit prokallikrein.

[0118] Table 55. Modified siRNAs and their sequences that inhibit prokallikrein.

[0119] Table 56. Modified siRNAs and their sequences that inhibit prokallikrein.

[0120] Table 57. Modified siRNAs and their sequences that inhibit prokallikrein.

[0121] Table 58. Modified siRNAs and their sequences that inhibit prokallikrein.

[0122] Table 59. Modified siRNAs and their sequences that inhibit prokallikrein.

[0123] Table 60. Modified siRNAs and their sequences that inhibit prokallikrein.

[0124] Example 3: A siRNA conjugate for inhibiting prokallikrein

[0125] This embodiment provides an siRNA conjugate for prokallikrein, the siRNA conjugate consisting of the modified siRNA of Example 2 and a GalNAc group conjugated to the modified siRNA, wherein the GalNAc group is attached to the 3' end of the positive strand of the modified siRNA; the siRNA conjugate has the following structure:

[0126] Add G after the name of the siRNA used in Example 2 to indicate the siRNA conjugate. For example, the siRNA conjugate corresponding to the modified siRNA PKK-74M1 is numbered PKK-74M1G.

[0127] Experimental Example 1: Detection of the on-target activity of siRNA and its modifications for inhibiting prokallikrein

[0128] This experimental example provides an on-target activity assay for siRNA and its modifications used to inhibit prokallikrein. The experimental procedure is as follows:

[0129] A single copy of the insert sequence (which is a fragment extracted from the full-length human KLKB1 mRNA sequence) was cloned into the Xho I / Not I site of the psiCHECKTM-2 plasmid (purchased from Suzhou Genecast Co., Ltd., catalog number C09005) to obtain the detection plasmids PKK1-psiCHECK2~PKK9-psiCHECK2 (insert sequences are shown in Table 61); the siRNA to be tested or the modified siRNA to be tested (for ease of description, the experimental process in this example is collectively referred to as siRNA) was diluted with DEPC water to obtain the siRNA dilution solution;

[0130] Add 5 μL of siRNA dilution buffer to each well of a 96-well plate. Add 12.5 μL of opti-MEM (Gibco, catalog number 31985-070) containing 20 ng of the detection plasmid to each well of a 96-well plate. Add 32.5 μL of opti-MEM to each well of a 96-well plate. Add 0.3 μL of Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, catalog number 11668-019) to each well of a 96-well plate. Let the plates stand at room temperature (25°C) for 15 min to obtain the mixed system (the detection plasmids corresponding to each siRNA are shown in Table 62).

[0131] After settling, it will contain 2×10 5DMEM complete medium (purchased from Transgen Biotech, catalog number FI101-01) containing 293T cells / mL (purchased from the Cell Bank of the Chinese Academy of Sciences) was added to 96-well plates at a rate of 50 μL / well, and the cells were cultured in a 5% (v / v) CO2 incubator at 37°C for 24 h (the final concentration of siRNA in the culture system was 0.1 nM). After the culture, the cells in the 96-well plates were subjected to dual-luciferase assay, and the results are shown in Tables 63 to 79 (each siRNA was tested in triplicate).

[0132] The dual-luciferase assay method is as follows:

[0133] Take the dual-luciferase assay kit (purchased from Promega, catalog number E2940), dilute the 5× lysis buffer in the kit with water to make 1× lysis buffer, and prepare substrate 1 and substrate 2 according to the kit instructions;

[0134] Take a 96-well plate, discard the supernatant, dilute each well with PBS buffer (purchased from Hyclone, catalog number SH30256.01) and wash twice, then add 1× lysis buffer at a rate of 50 μL / well to the 96-well plate and incubate at room temperature (25℃) for 20 min to lyse the cells in the 96-well plate to obtain the lysis buffer;

[0135] The lysis buffer was aspirated from the original 96-well plate and added to a new opaque 96-well plate at a rate of 30 μL / well. Substrate 1 and Substrate 2 were then added to the 96-well plate in portions of 30 μL / well / time. After each addition of substrate, the 96-well plate was analyzed using a multi-mode microplate reader to obtain the numerical results of firefly luciferase and Renilla luciferase, respectively.

[0136] The luminescence ratio was calculated for each well in a 96-well plate using the formula: Luminescence Ratio = Renilla luciferase value / Firefly luciferase value. The luminescence ratio for each test group (i.e., the group with the siRNA assay) or control group (i.e., the group with NC assay) was the average of the luminescence ratios from three replicates. Using the control group's luminescence ratio as a baseline, the luminescence ratios for each test group were normalized to obtain the luminescence ratio. (测试) / luminescence ratio (对照) The ratio R is used to represent the expression level (i.e., relative residual activity) of the Renal luciferase reporter gene. After obtaining the relative residual activity of the Renal luciferase reporter gene, the inhibition rate of siRNA on the KLKB1 gene is calculated according to the formula: inhibition rate = (1-R) ​​× 100%.

[0137] As shown in Tables 63-79, except for PKK-81M1, PKK-125M1, PKK-149M1, PKK-162M1~PKK-165M1, PKK-166M1, PKK-168M1, PKK-170M1, PKK-171M1, PKK-173M1, PKK-174M1, PKK-236M1, PKK-237M1, PKK-243M1, PKK-246M1, PKK-247M1, PKK-251M1, and PKK-291M1, the remaining modified siRNAs in Example 2 all showed resistance to PKK. The expression of mRNAs exhibited significant inhibitory activity; among them, PKK-4M1, PKK-16M1, PKK-17M1, PKK-22M1~PKK-35M1, PKK-52M1, PKK-54M1, PKK-56M1, PKK-60M1, PKK-62M1, PKK-65M1, PKK-71M1~PKK-75M1, PKK-77M1~PKK-80M1, PKK-82M1~PKK-88M1, PKK-90M1, PKK-103M1~PKK-114M1, PKK-1 PKK-22M1, PKK-141M1, PKK-176M1, PKK-186M1, PKK-187M1, PKK-188M1, PKK-190M1, PKK-191M1, PKK-192M1, PKK-193M1, PKK-197M1, PKK-209M1, PKK-215M1, PKK-222M1, PKK-223M1, PKK-233M1, PKK-260M1, PKK-262M1, PKK-276M1, and PKK-289M1 exhibit high inhibitory activity against PKK.

[0138] Table 61. Plasmid insertion sequence detection

[0139] Table 62. Detection plasmids corresponding to each siRNA to be tested

[0140] Table 63. Target activity of each siRNA to be tested

[0141] Table 64. Target activity of each siRNA to be tested

[0142] Table 65. Target activity of each siRNA to be tested

[0143] Table 66. Target activity of each siRNA to be tested

[0144] Table 67. Target activity of each siRNA to be tested

[0145] Table 68. Target activity of each siRNA to be tested

[0146] Table 69. Target activity of each siRNA to be tested

[0147] Table 70. Target activity of each siRNA to be tested

[0148] Table 71. Target activity of each siRNA to be tested

[0149] Table 72. Target activity of each siRNA to be tested

[0150] Table 73. Target activity of each siRNA to be tested

[0151] Table 74. Target activity of each siRNA to be tested

[0152] Table 75. Target activity of each siRNA to be tested

[0153] Table 76. Target activity of each siRNA to be tested

[0154] Table 77. Target activity of each siRNA to be tested

[0155] Table 78. Target activity of each siRNA to be tested

[0156] Table 79. Target activity of each siRNA to be tested

[0157] Experimental Example 2: Detection of the on-target activity of siRNA and its modifications for inhibiting prokallikrein

[0158] This experimental example provides an on-target activity assay for siRNA and its modifications used to inhibit prokallikrein. The experimental procedure is as follows:

[0159] 1. Construction of stable cell line hKLKB1-293T

[0160] The full-length sequence of human KLKB1 mRNA (GenBank ID NM_000892.5) was inserted into the LV5 vector to construct a lentiviral vector (provided by Suzhou Gemma Gene Co., Ltd.); the lentiviral vector and the packaging vector were transfected into 293T cells and cultured. The supernatant was collected to obtain hKLKB1 lentivirus (provided by Suzhou Gemma Gene Co., Ltd.).

[0161] 293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into MEM complete medium (purchased from Gibco, catalog number 11095-080) containing 10% (v / v) fetal bovine serum (FBS, purchased from Hyclone) and 0.2% (v / v) penicillin-streptomycin mixture (Penicillin-Streptomycin, purchased from Gibco, catalog number 15140122) and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h. After culture, the 293T cells were digested with trypsin (purchased from GIBCO, catalog number 25200-072). After digestion, the cells were rinsed with PBS buffer and then resuspended in MEM complete medium to obtain a cell concentration of 1×10⁶ cells / year. 5 Cell suspension of cells / mL;

[0162] Cell suspension was seeded into 24-well plates at a rate of 0.5 mL / well and cultured for 16 h in a 5% (v / v) CO2, 37°C cell culture incubator. After culture, hKLKB1 lentivirus was added to the 24-well plates at a multiplicity of infection (MOI) of 100, and the plates were cultured for another 72 h in a 5% (v / v) CO2, 37°C cell culture incubator to infect the 293T cells in the 24-well plates. After infection, the infected 293T cells were digested with trypsin. After digestion, the cells were first rinsed with PBS buffer and then resuspended in MEM complete medium containing 2 μg / mL puromycin. The 293T cells were resuspended after transformation. The resuspended cells were divided into 6 portions and passaged into 6-well plates. The cells were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 h for screening. After screening, the culture medium in the 6-well plates was replaced with an equal volume of MEM complete medium containing 1 μg / mL puromycin. The cells were then cultured in a 5% (v / v) CO2, 37°C cell culture incubator for another 24 h. After culture, the cultured cells were identified by real-time quantitative PCR. Successful identification indicates that the stable cell line hKLKB1-293T has successfully integrated the hKLKB1 gene at a high copy number.

[0163] 2. Detection of target activity

[0164] Step 1: RNA extraction

[0165] The siRNA to be tested or the modified siRNA to be tested (hereinafter referred to as siRNA in the experimental procedure description of this embodiment for ease of description) was diluted with opti-MEM to obtain the siRNA dilution solution; 25 μL of opti-MEM was mixed with 0.25 μL of Lipofectamine RNAiMAX transfection reagent to obtain the transfection reagent dilution solution; 25 μL of siRNA dilution solution containing different siRNAs was taken and mixed with the transfection reagent dilution solution respectively, and then incubated at room temperature (25℃) for 15 min to obtain the transfection solution containing siRNA;

[0166] Using transfection buffer without siRNA as a blank control (blank control group), transfection buffer containing 3×10⁻⁶ siRNA was used. 5 Cells of the stable hKLKB1-293T cell line were seeded into 96-well plates at a rate of 50 μL / well in MEM complete medium. Simultaneously, siRNA-containing transfection solution was seeded into the 96-well plates at a rate of 50 μL / well. The cells were then cultured in a 5% (v / v) CO2 incubator at 37°C for 48 h for transfection (the final concentration of siRNA in the transfection system was 0.1 nM or 1 nM). After transfection, the liquid in the wells was discarded, the cells were collected, and total RNA was extracted from the cells using a magnetic bead-based total RNA extraction kit (Germage-E31008-96).

[0167] Step 2: RNA extraction

[0168] Using HiScript III RT SuperMix for qPCR (purchased from Novizan, catalog number R323-01), a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions. Total RNA from the cells was reverse transcribed to obtain reverse transcription products (reverse transcription conditions: the reverse transcription reaction system was first incubated at 37°C for 15 min, then incubated at 85°C for 5 s); 80 μL of DEPC water was added to each reverse transcription product to obtain a solution containing cDNA.

[0169] Step 3: Preparation of qPCR reaction system

[0170] For each reverse transcription reaction system, take 4 μL of the above cDNA-containing solution as a template, and use the reagents provided in the AceQ Universal SYBR qPCR Master Mix kit (purchased from Vazyme, catalog number Q511-02) to prepare a 20 μL qPCR reaction system on an ice box according to Table 80 (in the qPCR reaction system, Primer1 and Primer2 are PCR primers for amplifying the target gene KLKB1 and the internal reference gene GAPDH, respectively; primer sequences are shown in Table 81); place each qPCR reaction system in an ABIStepOnePlus Real-Time... On a PCR instrument, a three-step amplification method was used to obtain product W containing the amplified target gene KLKB1 and the internal reference gene GAPDH (the amplification program of the qPCR reaction was: 95℃ pre-denaturation for 10 min, then 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s, repeating the above denaturation, annealing, and extension process for a total of 40 times); product W was then incubated sequentially at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. The melting curves of the target gene KLKB1 and the internal reference gene GAPDH in product W were collected by a real-time quantitative PCR instrument to obtain the Ct values ​​of the target gene KLKB1 and the internal reference gene GAPDH; the relative quantification of the target gene KLKB1 in each test group was calculated using the Ct (ΔΔCt) comparison method, and the calculation results are shown in Tables 53 to 55 (in the experiment, each siRNA to be tested was set up in 3 replicates).

[0171] The calculation method for the relative quantitative results is as follows:

[0172] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group);

[0173] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group);

[0174] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average);

[0175] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average);

[0176] Wherein, ΔCt (control group mean) is the arithmetic mean of ΔCt (control group) of the four samples in the control group; thus, each sample in the test group and the control group corresponds to a ΔCt value;

[0177] Using the control group as a baseline, the expression level of KLKB1 mRNA in the test group was normalized, and the expression level of KLKB1 mRNA in the control group was defined as 100%.

[0178] The relative expression level of KLKB1 mRNA in the test group was 2- ΔΔCt(测试组) ×100%;

[0179] The KLKB1 mRNA level was compared with the internal reference gene GAPDH, and the value was normalized to the mean of the saline control group. The data were expressed as a percentage relative to the saline control group and presented as the mean plus the standard deviation.

[0180] As shown in Tables 82-84, all modified siRNAs exhibit significant inhibitory activity against the expression of the prokallikrein-encoding gene (KLKB1 gene) in cells.

[0181] Table 80. RNA Amplification Reaction System

[0182] Table 81. Primer Information

[0183] Table 82. Target activity of each siRNA to be tested

[0184] Table 83. Target activity of each siRNA to be tested

[0185] Table 84. Target activity of each siRNA to be tested

[0186] Experimental Example 3: Detection of the on-target activity of siRNAs and their modifications used to inhibit prokallikrein

[0187] This experimental example provides an on-target activity assay for siRNA and its modifications used to inhibit prokallikrein. The experimental procedure is as follows:

[0188] Transgenic hKLKB1 mice (female, 6-8 weeks old, provided by Suzhou Genegene Co., Ltd.) expressing the full-length human KLKB1 mRNA sequence (GenBank ID: NM_000892.5) were divided into groups of 3 mice each. Each group of mice was given a single dose of 3 mg / kg of siRNA conjugate (the tested siRNA conjugates are shown in Table 83) or physiological saline (physiological saline was the control). On day 14 after administration, the mice were sacrificed, and liver samples were collected and flash-frozen in liquid nitrogen. mRNA was extracted from the mouse livers, and the target activity of the tested siRNA conjugates (the tested siRNA conjugates are shown in Table 85) was detected by RT-qPCR according to Example 2. The detection results are shown in Figure 1.

[0189] The steps for extracting liver mRNA are as follows:

[0190] Step 1: Take 20 mg of mouse liver tissue, add 1 mL of Trizol Lysis Buffer (purchased from Life Technology, catalog number 410701), grind and lyse the tissue to obtain the grinding product; transfer the grinding product to an RNase-free 1.5 mL centrifuge tube, shake for 15 s to fully lyse the tissue cells, and then let it stand at room temperature (25℃) for 5 min to obtain the lysate;

[0191] Step 2: Open the cap of the centrifuge tube, add 200 μL of chloroform (purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., catalog number 20140925) to the centrifuge tube, shake for 20 seconds, then let stand at room temperature (25℃) for 3 minutes, and then centrifuge at 4℃ and 12000×g for 20 minutes. After centrifugation, transfer the supernatant to a new 2.0 mL centrifuge tube, and add 1.5 times the volume of anhydrous ethanol (purchased from Jiangsu Qiangsheng Functional Chemical Co., Ltd., catalog number 20210802) to the centrifuge tube. Invert and mix well to obtain the mixture.

[0192] Step 3: Take a purification column with a collection tube (purchased from VWI, catalog number 11822AG0627), add 700 μL of the mixture obtained in Step 2 to the purification column, let it stand for 2 min, and then centrifuge the purification column at 4℃ and 10000×g for 1 min. After centrifugation, discard the filtrate; repeat the above steps for the remaining mixture.

[0193] Step 4: Add 700 μL of 80% (v / v) ethanol to the purification column, then centrifuge the column at 4°C and 10000×g for 1 min. After centrifugation, discard the filtrate.

[0194] Step 5: Add 700 μL of 80% (v / v) ethanol to the purification column, then centrifuge the purification column at 4℃ and 10000×g for 1 min. After centrifugation, discard the filtrate.

[0195] Step 6: Centrifuge the purification column at 10000×g for 2 min at 4℃. After centrifugation, remove the purification column with the collection tube (if there is liquid in the collection tube, please be careful not to splash the liquid onto the purification column), discard the collection tube, and put the purification column into a new 1.5 mL centrifuge tube; add 100 μL of DEPC water to the purification column, let it stand at room temperature (25℃) for 2 min, and then centrifuge at 10000×g for 1 min at 4℃. After centrifugation, collect the RNA solution for subsequent experiments.

[0196] As shown in Figure 1, PKK-74M1G, PKK-80M1G, PKK-110M1G, PKK-74M38G, PKK-80M38G and PKK-110M38G all have significant inhibitory activity against the expression of the prokallikrein-encoding gene (KLKB1 gene) in mice.

[0197] Table 85. Modified siRNAs and their sequences that inhibit prokallikrein.

[0198] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A siRNA for inhibiting prokallikrein, characterized in that, The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially anticomplementary to form a double-stranded region; the sense strand of the siRNA contains at least 15 consecutive nucleotides whose nucleotide sequences differ by no more than 3 nucleotides from any one of the nucleic acid molecules shown in any one of SEQ ID NO. 1 to 294; the antisense strand of the siRNA contains at least 15 consecutive nucleotides whose nucleotide sequences differ by no more than 3 nucleotides from any one of the nucleic acid molecules shown in any one of SEQ ID NO. 295 to 588.

2. The siRNA as described in claim 1, characterized in that, The nucleotide sequence of the sense strand of the siRNA is shown in any one of SEQ ID NO. 1 to 294; the nucleotide sequence of the antisense strand of the siRNA is shown in any one of SEQ ID NO. 295 to 588.

3. The siRNA as described in claim 1 or 2, characterized in that, At least one nucleotide in the sense or antisense strand of the siRNA is a modified nucleotide; the modification includes methoxy modification, fluorination modification, thiophosphate linkage and / or 2′-deoxynucleotide modification.

4. The siRNA as described in claim 3, characterized in that, The fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 6th, 14th, and 16th nucleotides of the antisense strand are fluorinated nucleotides; or, the fluorinated nucleotides are located in both the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the 7th, 8th, and 9th nucleotides of the sense strand are fluorinated nucleotides, and at least the 2nd, 10th, 12th, 14th, 16th, 18th, and 20th nucleotides of the antisense strand are fluorinated nucleotides.

5. The siRNA as described in claim 3, characterized in that, The methoxy-modified nucleotides are located in both the antisense and sense strands of the nucleotide sequence. Specifically, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 of the sense strand are methoxy-modified nucleotides, and at least the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, and 21 of the antisense strand are methoxy-modified nucleotides. The nucleotides are: or, the methoxylated nucleotides are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1, 2, 3, 4, 5, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 of the sense strand are methoxylated nucleotides, and at least the nucleotides at positions 1, 3, 4, 6, 8, 9, 11, 13, 15, 17, 19, and 21 of the antisense strand are replaced with methoxylated nucleotides.

6. The siRNA as described in claim 3, characterized in that, The nucleotides linked by the thiophosphate group are located in the antisense and sense strands of the nucleotide sequence, and, in the direction from the 5' end to the 3' end, at least the nucleotides at positions 1 and 2, and positions 2 and 3 of the sense strand are linked by thiophosphate groups, and at least the nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 of the antisense strand are linked by thiophosphate groups.

7. The siRNA as described in claim 3, characterized in that, The 2'-deoxynucleotide modified nucleotide is located in the antisense strand of the nucleotide sequence, and at least the 5th and 7th nucleotides of the antisense strand are 2'-deoxynucleotide modified nucleotides.

8. A siRNA conjugate, characterized in that, The siRNA conjugate contains the siRNA as described in any one of claims 1 to 7 and a conjugating group conjugated to the siRNA.

9. The siRNA conjugate as described in claim 8, characterized in that, The conjugating group is GalNAc.

10. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1 to 7.

11. A host cell, characterized in that, The host cell's genome is integrated with the siRNA according to any one of claims 1 to 7; or, the host cell carries the siRNA conjugate according to claim 8 or 9; or, the host cell carries the recombinant plasmid according to claim 10.

12. The use of the siRNA according to any one of claims 1 to 7, the siRNA conjugate according to claim 8 or 9, the recombinant plasmid according to claim 10, or the host cell according to claim 11 in the preparation of a medicament for the prevention and / or treatment of a disease, characterized in that, The disease is a prekallikrein-related disease, which is one or more of edema, swelling, thrombosis, stroke, or infarction.

13. The application as described in claim 12, characterized in that, The edema includes angioedema, macular edema, and / or cerebral edema; the thrombosis includes arterial embolism, venous embolism, and / or cerebral vascular embolism; the infarction includes myocardial infarction; and the stroke includes cerebrovascular stroke.

14. A prokaryotic release enzyme inhibitor, characterized in that, The inhibitor comprises the siRNA of any one of claims 1 to 7, the siRNA conjugate of claim 8 or 9, the recombinant plasmid of claim 10, and / or the host cell of claim 11.

15. A medicine for preventing and / or treating a disease, characterized in that, The disease is a disease related to prokallikrein; the drug comprises the siRNA of any one of claims 1 to 7, the siRNA conjugate of claim 8 or 9, the recombinant plasmid of claim 10, and / or the host cell of claim 11.

16. A method for preventing or treating diseases related to prokallikrein, characterized in that, This includes administering to a recipient the siRNA of any one of claims 1-7, the siRNA conjugate of claim 8 or 9, the recombinant plasmid of claim 10, the host cell of claim 11, the prokallikrein inhibitor of claim 14, or the drug of claim 15.

17. The method as described in claim 16, characterized in that, The dosage of the siRNA, siRNA conjugate, recombinant plasmid, host cell, prokallikrein inhibitor, or drug is 0.1-10 mg / kg.

18. The method as described in claim 16, characterized in that, The diseases associated with prokallikrein include vascular diseases and / or inflammatory diseases.

19. The method as described in claim 16, characterized in that, The diseases associated with prokallikrein include edema, thromboembolism, infarction, and / or stroke.

20. The method as described in claim 19, characterized in that, The edema includes angioedema, macular edema, and / or cerebral edema; the thromboembolism includes arterial embolism, venous embolism, and / or cerebral vascular embolism; the infarction includes myocardial infarction; and the stroke includes cerebrovascular stroke.

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