Small interfering rnas for zbp1 gene knockdown in live animals and uses thereof

Abstract

This invention provides a small interfering RNA (siRNA) for in vivo Zbp1 gene knockdown in animals and its application. The siRNA molecule comprises a sense strand and an antisense strand that complement each other to form a double-stranded region. The sense strand is 21 nt in length, and the antisense strand is 23 nt in length. Each nucleotide of both the sense and antisense strands is modified. The siRNA molecule is selected from: siRNA molecules with the sense strand nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand nucleotide sequence shown in SEQ ID NO: 2; and siRNA molecules with the sense strand nucleotide sequence shown in SEQ ID NO: 3 and the antisense strand nucleotide sequence shown in SEQ ID NO: 4. This siRNA molecule has a high knockdown efficiency for the Zbp1 gene, and this efficiency can be maintained for more than one week in continuously dividing cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a small interfering RNA for knocking down the Zbp1 gene in living animals and its applications. Background Technology

[0002] Mice are a key model organism for target validation in preclinical studies. Currently, methods for knocking down the Z-DNA binding protein 1 (Zbp1) gene in mice are limited to constructing Zbp1 knockout mice, which is not optimal for preclinical disease research. Small interfering RNA (siRNA) has been used in the treatment of human liver diseases; therefore, using siRNA to knock down Zbp1 in vivo is a promising approach. Zbp1 Genetic interference is feasible. However, the efficiency and persistence of RNA interference are affected by many factors. Ordinary small interfering RNA cannot work continuously in vivo and is prone to degradation. Summary of the Invention

[0003] To address the aforementioned issues, this invention proposes a small interfering RNA for knocking down the Zbp1 gene in living animals and its application. This RNA exhibits high knockdown efficiency for the Zbp1 gene, and this efficiency can be maintained for more than a week in continuously dividing cells.

[0004] To achieve the above objectives, the present invention provides, in a first aspect, a siRNA molecule for knocking down the Zbp1 gene in living animals, the siRNA molecule comprising a sense strand and an antisense strand that complement each other to form a double-stranded region, the sense strand being 21 nt in length and the antisense strand being 23 nt in length, each nucleotide of the sense strand and the antisense strand being modified.

[0005] The siRNA molecules are selected from:

[0006] The siRNA molecule with the sense strand nucleotide sequence as shown in SEQ ID NO: 1 and the antisense strand nucleotide sequence as shown in SEQ ID NO: 2;

[0007] The siRNA molecule has the nucleotide sequence of the sense strand as shown in SEQ ID NO: 3 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO: 4.

[0008] According to the present invention, a siRNA molecule for in vivo animal Zbp1 gene knockdown has a high knockdown efficiency for the Zbp1 gene, with a knockdown efficiency of over 90% after 36 hours of transfection; after modification, it still has an efficiency of nearly 90% after 72 hours of transfection, and this efficiency can be maintained for more than a week in continuously dividing cells, which is an effect that cannot be achieved by various current gene knockdown tools.

[0009] Optionally, the modification is selected from any one or more of the following:

[0010] 2'-Fluoro-modification (2'-fluoro, 2'-F), 2'-O-methyl-modification (2'-O-methyl, 2'-OMe), glycol nucleic acid (GNA), phosphorothioate (PS) linkage, vinyl phosphate group modification (5'-(E)-vinylphosphonate, 5'-(E)-VP), and 2'-16 alkyl oxygen modification (2'-O-C16).

[0011] Furthermore, the sense strand structure of the siRNA molecule is shown in (A1) or (A3), respectively; the antisense strand structure of the siRNA molecule is shown in (A2) or (A4), respectively.

[0012] (A1)ma·mc·mcmcmu(C16c)fAmafUfCfAmamgmumcmcmumumu·ma·ma(5'-3');

[0013] (A2) (5'-3');

[0014] (A3)mc·mc·mumgmu(C16a)fUmufCfCfAmumgmamgmamamamu·ma·ma(5'-3');

[0015] (A4) (5'-3');

[0016] Where ma, mc, mu, and mg represent nucleotides a, c, u, and g that have been modified with 2'-O-methyl, respectively; fA, fU, fC, and fG represent nucleotides A, U, C, and G that have been modified with 2'-fluorine, respectively; · indicates that the two nucleotides are linked by a phosphate thiophosphate backbone; Nucleotide G represents ethylene glycol-modified nucleic acid. denoted as ethylene glycol-modified nucleotide C; (C16c) represents nucleotide c modified with 2'-16 alkyl oxygen, (C16a) represents nucleotide a modified with 2'-16 alkyl oxygen; VP indicates modification of the 5' end with a vinyl phosphate group.

[0017] Furthermore, the chemical formula of the vinyl phosphate group is Formula I;

[0018]

[0019] Furthermore, the chemical formula of the 2'-16 alkyl oxygen is Formula II;

[0020]

[0021] In a second aspect, the present invention provides a mixture of RNAi molecules for knocking down the Zbp1 gene in living animals, comprising the aforementioned siRNA molecules.

[0022] In a third aspect, the present invention proposes the application of the above-mentioned siRNA molecule or the above-mentioned RNAi molecule mixture in the preparation of biomaterials for knocking down the Zbp1 gene in living animals.

[0023] Optionally, the animal is a mouse.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0025] Figure 1 This is a diagram showing the effect of the siRNA sequence of Example 1 of the present invention on the knockdown of the Zbp1 gene;

[0026] Figure 2 This is a diagram showing the effect of the siRNA sequence in Example 2 of the present invention on the knockdown of the Zbp1 gene. Detailed Implementation

[0027] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0028] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0029] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0030] Example 1: In vitro screening of siRNA sequences targeting the Zbp1 gene (cellular level)

[0031] In this embodiment, Ripk1 KO L929 cells (PMID: 28176780) treated with 1 μg / mL IFN-γ for 24 h were selected for in vitro screening of target siRNA sequences.

[0032] The siRNA in this example:

[0033] siNC is a negative control sequence without specific targeting (provided by Guangzhou Ruibo Biotechnology Co., Ltd., product number siN0000001-1-5);

[0034] siAct is a positive control sequence targeting the housekeeping gene Actin (provided by Guangzhou Ruibo Biotechnology Co., Ltd., product number siP0000003-1-5);

[0035] siZ1 is an unmodified Zbp1 siRNA sequence (customized by the applicant from Guangzhou Ruibo Biotechnology Co., Ltd., product number siG141125145050, target sequence is CCCTCAATCAAGTCCTTTA);

[0036] siZ2 is an unmodified Zbp1 siRNA sequence (customized by the applicant from Guangzhou Ruibo Biotechnology Co., Ltd., product number siG141125145103, target sequence is GCCTGCAACATGGAGCATA);

[0037] siZ3 is an unmodified Zbp1 siRNA (custom-made by the applicant from Guangzhou Ruibo Biotechnology Co., Ltd., product number siG141125145118, target sequence is CCTGTATTCCATGAGAAAT).

[0038] After transfecting L929 cells with JinBaoLuo-siRNA Plus (Xiamen JinBaoLuo Technology Co., Ltd., catalog number A003) for 36 hours, the cells were lysed to obtain protein lysis buffer. Specifically, protein lysis buffer was obtained by transfecting L929 cells with JinBaoLuo-siRNA Plus for 5 × 10⁶ cells per cell. 5Add a mixture containing 3 μL JinBaoLuo-siRNA Plus transfection reagent and 4 μL 20 μM siRNA to each L929 cell. After transfection at room temperature, incubate at 37°C for 12 h, then replace with fresh DMEM medium. 36 h after transfection, remove the medium and directly mix the cells in one well of a 12-well plate with 80 μL 2×SDS sample buffer. Collect the cells to obtain protein lysis buffer, collect this lysis buffer into a 1.5 mL tube, boil in a metal bath at 100°C for 10 min, then mix and centrifuge. This sample is ready for subsequent Western blot analysis.

[0039] Western blotting was used to detect protein expression levels: GAPDH was used as the internal control protein, and the primary antibody was derived from ABclonal (catalog number AC002), with a band size of approximately 36 kDa. Mouse ZBP1 has long and short isoforms, with sizes of approximately 60 kDa and 25 kDa, respectively; the primary antibody was derived from AdipoGen (catalog number AG-20B-0010-C100). The secondary antibody for both was goat anti-mouse IgG (from Thermo Fisher Scientific, catalog number 31430). Due to the significant difference in molecular weight between the two target proteins, therefore... Figure 1 After completing the primary antibody-secondary antibody-exposure process on ZBP1, the Western blot was repeated on the same PVDF membrane using the primary antibody-secondary antibody-exposure process for GAPDH.

[0040] The results are as follows Figure 1 As shown, 36 hours after transfection, over 90% of the Zbp1 long isoform was knocked down by siZ1, and nearly 100% of the Zbp1 short isoform was knocked down by siZ1; the knockdown effect of siZ3 was the second best. Therefore, siZ1 and siZ3 were selected for further optimization experiments.

[0041] Example 2

[0042] The siZ1 and siZ3 sequences of Example 1 are modified so that both the siZ1 and siZ3 sequences are composed of a sense chain and an antisense chain;

[0043] The justice chain of siZ1: 5'-acccucAaUCAaguccuuuaa-3' (SEQ ID NO: 1);

[0044] The antisense chain of siZ1: (SEQ ID NO: 2);

[0045] The justice chain of siZ3: 5'-ccuguaUuCCAugagaaauaa-3' (SEQ ID NO: 3);

[0046] The antisense chain of siZ3: (SEQ ID NO: 4);

[0047] The modification strategies are as follows: (1) The entire chain needs to be modified by ESC: uppercase letters are fluorinated (2'-F), lowercase letters are modified with methoxy (2'-OMe), and the bold underlined letters G or C on the antisense chain are ethylene glycol nucleic acid (GNA) modifications; (2) A vinyl phosphate group 5'-(E)-VP needs to be added to the 5' end of the antisense chain, with the chemical formula shown in Formula I; (3) A 16 alkyl oxygen (2'-O-C16) needs to be added to the sense chain, with the chemical formula shown in Formula II; (4) PS linkage (phosphorothioate linkage) needs to be added between the three nucleotides at the 5' end of the sense chain and between the three nucleotides at the 3' end of the antisense chain.

[0048]

[0049] The modified siZ1 and siZ3 are named siZ1*(#1-siZbp1) and siZ3*(#3-siZbp1), respectively, and their nucleotide compositions are as follows:

[0050] The chain of justice of siZ1*: ma·mc·mcmcmu(C16c)fAmafUfCfAmamgmumcmcmumumu·ma·ma(sense 5'-3');

[0051] The antispeech chain of siZ1*: (antisense 5'-3');

[0052] The chain of justice of siZ3*: mc·mc·mumgmu(C16a)fUmufCfCfAmumgmamgmamamamu·ma·ma(sense 5'-3');

[0053] The antispeech chain of siZ3*: (antisense 5'-3');

[0054] In the above, ma, mc, mu, and mg represent nucleotides a, c, u, and g modified by 2'-OMe, respectively; fA, fC, fU, and fG represent nucleotides A, C, U, and G modified by 2'-F, respectively; · indicates that the two nucleotides are linked by a PS linker; (C16c) and (C16a) represent nucleotides c and a modified by 2'-O-C16, respectively; VP indicates 5'-(E)-VP modification; and the bold underline indicates GNA modification. The designed siZ1* and siZ3* were synthesized by Jima Gene Technology Co., Ltd.

[0055] Similar to the cell transfection method in Example 1, L929 cells were transfected with siZ1* and siZ3* for 24 hours, 72 hours, or 7 days. The cells were then lysed to obtain protein lysates, and the protein expression levels were detected by Western blot.

[0056] The results are as follows Figure 2 As shown, 24 hours post-transfection, siZ1, siZ1*, and siZ3* all knocked down the two isoforms (long and short) of ZBP1 with approximately 100% efficiency. At 72 hours post-transfection, with cell division and passage, the modified siRNAs (siZ1* and siZ3*) still knocked down the two isoforms of ZBP1 with approximately 90% efficiency, while the unmodified siRNA had a knockdown efficiency of approximately 75%. With continued passage, on day 7 post-transfection, all three siRNAs showed no significant knockdown efficiency against the ZBP1 short variant; the unmodified siRNA had a knockdown efficiency of only 30-40%, siZ1* had a 60% knockdown efficiency, and siZ3* still had at least 80% knockdown efficiency. Therefore, the modification and optimization of siZ1* and siZ3* effectively achieved persistent knockdown of ZBP1 expression.

[0057] In summary, according to embodiments of the present invention, siRNA modification achieves a high knockdown efficiency for the Zbp1 gene, and this efficiency can be maintained for more than a week in continuously dividing cells, which is an effect that cannot be achieved by current gene knockdown tools.

[0058] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0059] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A device for use on live animals Zbp1 Gene knockdown siRNA molecules are characterized by, The siRNA molecule comprises a sense strand and an antisense strand that complement each other to form a double-stranded region. The sense strand is 21 nt long and the antisense strand is 23 nt long. Each nucleotide of the sense strand and the antisense strand is modified. The siRNA molecules are selected from: The siRNA molecule has the sense strand nucleotide sequence shown in SEQ ID NO: 1 and the antisense strand nucleotide sequence shown in SEQ ID NO: 2; the sense strand structure of the siRNA molecule is shown in (A1), and the antisense strand structure of the siRNA molecule is shown in (A2); Or an siRNA molecule with the sense strand nucleotide sequence as shown in SEQ ID NO: 3 and the antisense strand nucleotide sequence as shown in SEQ ID NO: 4; the sense strand structure of the siRNA molecule is shown in (A3); the antisense strand structure of the siRNA molecule is shown in (A4); (A1)ma·mc·mcmcmu(C16c)fAmafUfCfAmamgmumcmcmumumu·ma·ma (5'-3'); (A2)VP-mu·fU·mamamg G mamcmumumgmafUmufGmamgmgmgmu·mu·mu (5'-3'); (A3) mc·mc·mumgmu(C16a)fUmufCfCfAmumgmamgmamamamu·ma·ma (5'-3'); (A4)VP-mu·fU·mamumu C mumcmamumgmgfAmafUmamcmamgmg·ma·mg (5'-3'); Where ma, mc, mu, and mg represent nucleotides a, c, u, and g that have been modified with 2'-O-methyl, respectively; fA, fU, fC, and fG represent nucleotides A, U, C, and G that have been modified with 2'-fluorine, respectively; · indicates that the two nucleotides are linked by a phosphate thiophosphate backbone; G Nucleotide G represents ethylene glycol-modified nucleic acid. C denoted as ethylene glycol-modified nucleotide C; (C16c) represents nucleotide c modified with 2'-16 alkyl oxygen, (C16a) represents nucleotide a modified with 2'-16 alkyl oxygen; VP indicates modification of the 5' end with a vinyl phosphate group.

2. The method for use on live animals as described in claim 1 Zbp1 Gene knockdown siRNA molecules are characterized by, The chemical formula of the vinyl phosphate group is Formula I; Formula I.

3. The method for use on live animals as described in claim 1 Zbp1 Gene knockdown siRNA molecules are characterized by, The chemical formula of the 2'-16 alkyl oxygen is Formula II; Formula II.

4. A device for use on live animals Zbp1 A mixture of gene knockdown RNAi molecules, characterized in that, It contains the siRNA molecule as described in any one of claims 1-3.

5. The siRNA molecule as described in any one of claims 1-3 or the RNAi molecule mixture as described in claim 4 in the preparation of knockdown animals in vivo. Zbp1 Application of genes in biological materials, wherein the animal is a mouse.

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