siRNAs that inhibit IL-25 gene expression and their applications

Abstract

This invention discloses an siRNA that inhibits IL-25 gene expression and its application. The siRNA sequence provided by this invention can specifically knock down IL-25 expression levels. Furthermore, by modifying the sense strand with thiocyanate, this invention enhances the ability of siRNA to resist enzymatic hydrolysis in serum and prolongs its duration of action without significantly increasing cytotoxicity, thus bringing important application value to the treatment of autoimmune diseases.

Description

Technical Field

[0001] This invention relates to an siRNA that inhibits IL-25 gene expression and its application, belonging to the field of biomedical technology. Background Technology

[0002] IL-25 is a member of the interleukin-17 family, also known as IL-17E. Human IL-25 is 3987 bp in length and located on chromosome 14. Its receptor is IL-17RB. IL-25 primarily acts on non-B / non-T cells that highly express MHC-II and low CD11c; on the T cell side, it targets Th2 memory cells, NKT cells, and CD14 cells. + Cells are also target cells of IL-25. Upon stimulation by IL-25, NF-κB activator 1 binds to IL-17RB via its intracellular SEF1R domain, activating the NF-κB signaling pathway. IL-25 can also promote IL-17RB expression and increase the function of Th2 memory cells through the interaction between thymic stromal lymphopoietin (TSLP) and dendritic cells (DCs), thus playing an immunomodulatory role. Existing research indicates that IL-25 is involved in at least the following immune diseases: psoriasis, allergic pneumonia, rheumatoid arthritis, allergic granulomatous vasculitis, and autoimmune diabetes. Therefore, IL-25 is a potential therapeutic target for autoimmune diseases.

[0003] The mechanism of action of siRNA is also known as RNA interference or RNAi. siRNA is typically a 19-25 bp double-stranded RNA molecule. Its function is to induce efficient and specific degradation of homologous mRNA, thereby reducing the expression of a specific protein. Intracellular dsRNA is recognized by the Dicer enzyme (a member of the RNase III family) and cleaved into siRNA fragments. The siRNA then forms an RNA-mediated RISC complex with other intracellular proteins. This complex seeks out mRNA homologous to the antisense strand of the siRNA. Under the action of ATP, the Dicer enzyme unwinds the siRNA, releasing the sense strand while retaining the antisense strand. The antisense strand specifically pairs with the mRNA, and the RISC rapidly cleaves this mRNA. Because this mRNA lacks the protection of a poly(A) tail and a 5' cap, it can be rapidly hydrolyzed by other nucleases, thereby inhibiting the translation of this mRNA into a protein. Therefore, siRNA specifically reduces the expression of mRNA and even protein at the protein level.

[0004] There are two major challenges to the application of siRNA in the biomedical field. One is sequence specificity, and the other is its susceptibility to degradation by nucleases. While siRNA sequences can tolerate 1-2 base mismatches with mRNA, the probability of targeting other non-target mRNAs increases significantly when 3 or more mismatches occur. Furthermore, the interference efficiency of siRNA may decrease with the presence of mismatched bases. Therefore, designing a highly specific siRNA is the first problem to solve. Secondly, when siRNA is delivered in vitro into the body, it is easily degraded and metabolized due to the abundance of nucleases in the body, or it may produce undesirable immune stimuli. Chemical modification of the siRNA sequence can improve its stability and reduce negative immune stimuli. Commonly used chemical modifications include phosphate backbone modification, ribose modification, and base modification. For example, patent number CN 107904239 A verified that unmodified siRNA-RB was significantly degraded after 30 minutes, while chemically modified siRNA-RB showed no significant degradation within 30 minutes. Designing effective siRNA modifications that do not affect knockdown levels is another problem to be solved.

[0005] Currently, in the RNAi field, several siRNA drugs approved by the FDA belong to Anlylam, meaning Anlylam holds a significant share of the siRNA drug market and has a rapid pace of siRNA drug development. The first drug to be marketed was Patisiran, approved by the FDA in August 2018 for the treatment of nerve damage caused by transthyretin amyloidosis (haTTR). Both the sense and antisense strands contain 21 nucleotides. The 19 nucleotides of the sense strand hybridize with the complementary 19 nucleotides of the antisense strand, forming 19 nucleotide base pairs, leaving two 3'-terminal nucleotides on each strand as unhybridized overhangs. Patisiran also utilizes 2'-OM and 2'-O-Me modifications. Subsequent drugs marketed include givasiran for treating acute hepatic porphyria and Lumasilan for treating primary type 1 hyperoxaluria. Currently, domestic company Shengnuo Pharmaceuticals is accelerating the development of an siRNA drug for treating hypertrophic scars, which has entered Phase II clinical trials. Currently, there are no reports on the development of siRNA drugs for treating autoimmune diseases such as psoriasis. This invention designs and synthesizes an siRNA sequence based on the gene sequence characteristics of IL-25, aiming to specifically knock down the expression level of the IL-25 gene. Simultaneously, the sequence is modified to address the stability issues of siRNA in serum and avoid potential cytotoxicity, thereby achieving the goal of effectively improving and treating autoimmune diseases. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention obtains a pair of siRNAs (target gene IDs: NM_172314.2 and NM_022789.4) that specifically knock down IL-25 expression and function through screening. The sense and antisense strands are shown as SEQ ID No.1 and SEQ ID No.2, respectively, and are suitable for in vivo and in vitro experiments.

[0007] The first objective of this invention is to provide an siRNA that inhibits IL-25 gene expression, wherein the sequence of the sense strand of the siRNA is 5'-CCUGGAGAUAUGAGUUGGATT-3' and the sequence of the antisense strand is 3'-TTGGACCUCUAUACUCAACCU-5'.

[0008] Furthermore, the siRNA specifically targets the corresponding mRNA homologous sequence fragment of the IL-25 gene, which is numbered NM_172314.2 or NM_022789.4 by NCBI.

[0009] Furthermore, the siRNA also includes chemical modification of its sense strand.

[0010] Furthermore, the chemical modification is a thiomodification.

[0011] Furthermore, the thiomodification involves replacing one oxygen atom of the α-phosphate group in the bases at positions 2, 3, 4, 5, 7, 9, 10, 11, 12, 14, 16, 17, 18, and 20 of the positive chain with a sulfur atom.

[0012] Furthermore, the thiomodification involves replacing one oxygen atom of the α-phosphate group in the bases at positions 2, 3, 4, 7, 9, 10, 11, 12, 14, 16, 17, and 20 of the positive chain with a sulfur atom.

[0013] A second objective of this invention is to provide the use of the siRNA in the preparation of medicaments for treating immune diseases.

[0014] Furthermore, the aforementioned immune diseases include at least one of psoriasis, ankylosing spondylitis, and systemic lupus erythematosus.

[0015] Furthermore, the drug is administered via skin, intravenous injection, or mucosal administration.

[0016] Furthermore, the drug is delivered into the body via a delivery carrier, such as a liposome (LNP), through skin administration (subcutaneous injection, intradermal injection), mucosal administration (nasal atomization), and systemic administration (intravenous drip), to exert its effect and treat autoimmune diseases.

[0017] The beneficial effects of this invention are:

[0018] The siRNA sequence provided by this invention can specifically knock down the expression level of IL-25. Furthermore, by modifying the sense strand with thiocyanate, this invention improves the ability of siRNA to resist enzymatic hydrolysis in serum and prolongs its duration of action without significantly increasing cytotoxicity, thus bringing important application value to the treatment of autoimmune diseases. Attached image description:

[0019] Figure 1 It is a thiolated structural formula;

[0020] Figure 2 The purified product obtained after preparing siIL-25 is subjected to quality inspection;

[0021] Figure 3 This is the process of screening SEQ ID No. 1 and SEQ ID No. 2 using IL-25 siRNA;

[0022] Figure 4 This is a verification of the anti-enzymatic ability of 3 pairs of siIL-25 in serum;

[0023] Figure 5 It is the inhibitory effect of two pairs of chemically modified siIL-25 on the mRNA level of target genes;

[0024] Figure 6 It is the inhibitory effect of two pairs of chemically modified siIL-25 on the target gene protein level;

[0025] Figure 7 The effects of two chemically modified siIL-25s on cytotoxicity. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0027] Example 1: Screening for siRNAs that effectively knock down IL-25 mRNA

[0028] (1) Design three pairs of IL-25 siRNAs, abbreviated as siIL25-1, siIL25-2, and siIL25-3. The sequences were compared in the NCBI database. They are mRNAs that specifically target IL-25 and do not bind to other non-target genes.

[0029] (2) The crude product of siIL-25 was obtained by solid-phase synthesis using a nucleic acid synthesizer. The crude product was purified by AKTAexplorer100 to obtain the final product.

[0030] (3) Taking siIL25-2 as an example, Figure 2 A is the chromatogram of siIL25-2 during purification. The product in the '1-A2' region of the chromatogram is collected as the purified product. Figure 2 B is the purified product. During liquid chromatography, siRNA eluted around 13.871 min, with a purity greater than 95%. A small amount of siIL25-2 was then subjected to agarose gel electrophoresis to obtain... Figure 2 In the middle C, 1 and 2 are the positions of the siRNA product, and the product size is about 21 bp.

[0031] (4) Transfected cells

[0032] ① Quantify siRNA, dilute with DEPC water to 20uM.

[0033] ②HaCat cells were passaged into 12-well plates, and the density reached 30%–50% before transfection on the second day.

[0034] ③ During transfection, add 2.5 μL siRNA to 50 μL DMEM medium as tube A, and add 5 μL lip3000 to 50 μL DMEM medium as tube B. Add the contents of tube A to tube B, mix thoroughly, and let stand at room temperature for 15 min. Pour the mixture into a 12-well plate. Set up a control (NC) group, and perform three replicates for each experimental group.

[0035] ④ At 48h, RNA genome was extracted using the Tiangen 'RNA prep Pure Cell / Bacteria Kit', catalog number DP430.

[0036] ⑤ Reverse transcription yields cDNA.

[0037] ⑥Q-PCR was used to detect the mRNA expression level of IL-25.

[0038] (5) Experimental Results

[0039] Figure 3 The results are from HaCat cells transfected with siIL25-1, siIL25-2, and siIL25-3. Figure 3 According to our analysis, all three siRNA sequences we designed effectively knocked out IL-25 upon transfection with 50 pmol, with siIL25-2 showing the best mRNA knockdown effect. Figure 3In step B, we found that when we transfected siIL25-2 at 80 pmol, the knockdown effect on IL-25 was still significant. Therefore, we selected siIL25-2 for further research. siIL25-2 is SEQ ID No. 1 (5'-CCUGGAGAUAUGAGUUGGATT-3') and SEQ ID No. 2 (3'-TTGGACCUCUAUACUCAACCU-5') in this invention. The NCBI numbers of the mRNA sequences targeted by SEQ ID No. 1 and SEQ ID No. 2 are NM_172314.2 or NM_022789.4. The siIL25 mentioned below refers to the siIL25-2 sequence pair.

[0040] Example 2: Chemical modification method for screening siIL-25

[0041] (1) Design two pairs of chemical modification methods for siIL-25, named siIL-25mode1 and siIL-25mode2. The sequence of siIL-25mode1 is: 5'-C*C*U*G*GA*GA*U*A*U*GA*GU*U*G*GA*TT-3', antisense chain: 3'-TTGGACCUCUAUACUCAACCU-5'; the sequence of siIL-25mode2 is: 5'-C*C*U*GGA*GA*U*A*U*GA*GU*U*GGA*TT-3', antisense chain: 3'-TTGGACCUCUAUACUCAACCU-5'. The position of the thio group in the phosphodiester bond and the thio group's chemical structure are shown below. Figure 1 As shown, Figure 1 In the middle, A represents the position of the thioglycolate in the RNA chain. Figure 1 B is the chemical structural formula for thiolation.

[0042] Table 1

[0043] name sequence SEQ ID No.1 5'-CCUGGAGAUAUGAGUUGGATT-3' SEQ ID No.2 3'-TTGGACCUCUAUACUCAACCU-5' mode1 5'-C*C*U*G*GA*GA*U*A*U*GA*GU*U*G*GA*TT-3' mode2 5'-C*C*U*GGA*GA*U*A*U*GA*GU*U*GGA*TT-3'

[0044] (2) Serum stability verification

[0045] ① Take multiple 0.2ml PCR tubes, add 800ng siIL-25mode1 and 1ul fetal bovine serum respectively, and make up to 10ul with DEPC water. Incubate at 37℃ for N hours, N = 6h, 4h, 2h, and 0h respectively. The procedure for siIL-25mode2 is the same.

[0046] ②The procedure for unmodified siIL-25 is the same as above.

[0047] ③ Run agarose gel electrophoresis at 140V for 15 minutes.

[0048] ③ Results of serum stability test:

[0049] Figure 4 In the diagram, 1, 2, 3, and 4 represent the positions of the siIL-25mode1 band, 5, 6, 7, and 8 represent the positions of the siIL-25mode2 band, 9, 10, 11, and 12 represent the positions of the siIL25 band (siIL25-2: SEQ ID No. 1 and SEQ ID No. 2), 13 represents the position of xylene cyanine, and 14 represents the position of bromophenol blue; 1, 5, and 9 represent 0h, 2, 6, and 10 represent 2h, 3, 7, and 11 represent 4h, and 4, 8, and 12 represent 6h.

[0050] Depend on Figure 4 The results showed that siIL-25mode1 and siIL-25mode2 were gradually degraded in serum over time. At 6 h, there was still obvious siRNA present in siIL25. In contrast, siIL-25 was expected to degrade most of the siRNA at 2 h and almost all of it at 4 h. This indicates that the chemically modified siIL-25mode1 and siIL-25mode2 improved the enzymatic hydrolysis ability.

[0051] (3) mRNA level verification

[0052] ① Following the method in Example 1, siIL-25mode1 and siIL-25mode2 were purified, transfected into HaCat cells, and cellular RNA was extracted, reverse transcribed into cDNA, and IL-25 expression was measured by Q-PCR.

[0053] ② mRNA experimental results

[0054] Depend on Figure 5 The left and middle figures show that mode1 exhibits good knockdown effects in the range of 75 pmol to 100 pmol, with approximately 75% mRNA knockdown at 75 pmol. Figure 5 As shown in the middle right figure, mode2 has a good knockdown effect in the range of 50 pmol to 100 pmol, with about 90% of mRNA knocked down at 100 pmol.

[0055] (4) Protein level verification

[0056] ① Following the method in Example 1, siIL-25mode1 and siIL-25mode2 were purified and then transfected into HaCat cells.

[0057] ② After 48 hours, wash the cells once with PBS, add 200 μL of cell lysis buffer (RIPA) containing protease inhibitors, pipette thoroughly and transfer to a 1.5 ml EP tube; centrifuge at 12,000 rpm for 5 min at room temperature, and collect the supernatant, which is the total cell protein product.

[0058] ③ Place in a water bath at 95℃ for 5 minutes to fully denature the protein. After quantification, store at -80℃ for later use.

[0059] ④ Prepare polyacrylamide gels: 8% stacking gel and 12% separating gel. Load protein samples at 60V for 30 min and 120V for 90 min.

[0060] ⑤ Cut a PDVF membrane of appropriate size, activate it with methanol for 20 seconds, discard the methanol, wash with pure water for 5 minutes, disassemble the gel casting machine, place it in the membrane washing box, discard the water, pour in the transfer solution, and wash on a shaker for 15 minutes.

[0061] ⑥ Place the white side of the transfer film on the white side and the glue on the black side. Place the glue in the middle layer of cotton. Generally, there are two layers of cotton, glue, and two more layers of cotton. Cut a notch in the upper left corner of the film as a mark.

[0062] ⑦ Place it in the transfer tank, put it in a basin, fill the tank with transfer solution, cover it, pour in ice to fill the tank, add an appropriate amount of water (the water should not exceed the lid to prevent unstable current), 100V, 2h.

[0063] ⑧ Dilute 5% skim milk powder with TBST, immerse the membrane in 5% milk, and seal it by shaking for 2 hours.

[0064] ⑨ Rinse the membrane briefly in TBST, dilute the primary antibody with the primary antibody diluent, apply it to the membrane, and incubate overnight at room temperature.

[0065] ⑩ Wash the membrane with TBST + 0.1% Tween-20 at room temperature for 10 minutes, 3 times.

[0066] Dilute the secondary antibody with the secondary antibody dilution buffer, apply to a membrane, and incubate at room temperature for 2 hours.

[0067] Wash the membrane with TBST for 5 min at room temperature, 4 times; wash with TBS for 5 min at room temperature, once; develop in a darkroom.

[0068] Protein experiment results

[0069] Figure 6 A and B in the figure are the results of Western blot experiments. Figure 6In A, 1 and 7 represent the position of IL-25 protein at 20 kDa, 2 and 8 represent the position of GAPDH at 35 kDa, 3 represents the siNC 50 pmol group, 4 represents the mode1 50 pmol group, 5 represents the mode2 50 pmol group, 6 and 12 represent the cell blank control group, 9 represents the siNC 75 pmol group, 10 represents the mode1 75 pmol group, and 11 represents the mode2 75 pmol group. Figure 6 Figure B is a grayscale statistical graph. The experimental results show that when transfected with mode1 at 50 pmol, the IL-25 protein level decreased; when transfected with mode1 at 75 pmol or mode2 at 75 pmol, the IL-25 protein level decreased significantly. This indicates that mode1 and mode2 effectively reduced the expression of IL-25 protein.

[0070] (5) Some invalid chemical modification sequences

[0071] When screening chemical modification methods, we found that many sequences chemically modified for siIL-25 lacked resistance to serum enzyme hydrolysis or failed to effectively knock down IL-25 mRNA levels. This list only includes a small subset of chemical modification methods that are ineffective against IL-25.

[0072] Table 2

[0073]

[0074] Example 3: Cytotoxicity detection of siIL-25mode1 and siIL-25mode2

[0075] (1) Some literature reports that thiomodification of siRNA may increase cytotoxicity, and the more thiomodified bases there are, the greater the probability of cytotoxicity. We used HaCat cells (human immortalized keratinocytes) to verify the cytotoxicity of the chemically modified siIL-25 of this invention.

[0076] ① On the first day, cells were passaged into 96-well plates so that the cell density was 50% when siRNA was added on the second day.

[0077] ② On the second day, add 500 pmol of siIL-25mode1 or siIL-25mode2 to each well as needed for the experiment.

[0078] ③Incubate overnight at 37°C with 5% CO2.

[0079] ④ The kit is used to detect cell viability, and the absorbance is measured at 450 nm using an ELISA reader.

[0080] (2) Experimental Results

[0081] Depend on Figure 7It was found that mode1 and mode2 did not significantly affect cell viability at 2h, 4h, 6h, 16h, and 24h, and were essentially non-toxic.

[0082] In summary, the unmodified siIL-25 provided by this invention effectively knocks down IL-25 mRNA levels. After chemical modification, it not only effectively reduces the expression levels of both mRNA and protein but also significantly enhances resistance to enzymatic degradation in serum, with virtually no cytotoxicity. The sequence provided by this invention can be used in in vitro and in vivo experiments. For autoimmune diseases, the sequence provided by this invention offers significant application value for research on innovative biological drugs and small molecule nucleic acid drugs.

[0083] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A siRNA that inhibits IL-25 gene expression, characterized in that, The sequence of the sense strand of the siRNA is 5'-CCUGGAGAUAUGAGUUGGATT-3', and the sequence of the antisense strand is 3'-TTGGACCUCUAUACUCAACCU-5'.

2. The siRNA according to claim 1, characterized in that, The siRNA also includes chemical modification of its positive strand.

3. The siRNA according to claim 2, characterized in that, The chemical modification is a thiomodification.

4. The siRNA according to claim 3, characterized in that, The thiomodification involves replacing one oxygen atom in the α-phosphate group of the bases at positions 2, 3, 4, 5, 7, 9, 10, 11, 12, 14, 16, 17, 18, and 20 of the positive chain with a sulfur atom.

5. The siRNA according to claim 3, characterized in that, The thiomodification involves replacing one oxygen atom in the α-phosphate group of the bases at positions 2, 3, 4, 7, 9, 10, 11, 12, 14, 16, 17, and 20 of the positive chain with a sulfur atom.

6. The use of the siRNA according to any one of claims 1 to 5 in the preparation of a medicament for treating immune diseases, characterized in that, The aforementioned immune disease is psoriasis.

7. The application according to claim 6, characterized in that, The drug is administered via skin, intravenous injection, or mucosal administration.

8. The application according to claim 7, characterized in that, The skin administration includes subcutaneous injection or intradermal injection, and the mucosal administration includes nasal inhalation via nebulization.

Images