SiRNA for inhibiting CD47 gene expression and application thereof
By designing a siRNA sequence targeting CD47, RNA interference therapy is used to reduce the CD47 expression level, the problem of accidental damage to red blood cells by existing CD47-targeted drugs is solved, and efficient and safe tumor treatment effect is achieved.
Patent Information
- Application Number
- CN202411791251.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-14
AI Technical Summary
The existing CD47-targeted drugs kill tumor cells while being unable to avoid accidentally damaging red blood cells, resulting in difficulty in ensuring safety and reducing efficacy.
A series of small interfering RNA (siRNA) sequences were designed to target the inhibition of CD47 gene expression, and the expression level of CD47 was reduced through RNA interference therapy to avoid the impact on red blood cells.
At lower concentrations, siRNA can significantly reduce the CD47 expression level to more than 95%, and the inhibitory efficiency of more than 90% is maintained at a dosing concentration of 1 nM, reducing the side effects on red blood cells and improving the safety of treatment.
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Figure CN119614567A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine and relates to siRNA for inhibiting CD47 gene expression and application thereof. Background Art
[0002] CD47, also known as integrin-associated protein (IAP), is a cell membrane protein belonging to the immunoglobulin superfamily. It is widely expressed on the surface of almost all normal cells and highly expressed in tumor cells. Studies have shown that CD47 is highly expressed in a variety of hematological tumors and solid tumors, including primary effusion lymphoma, pancreatic ductal adenocarcinoma, non-Hodgkin's lymphoma, acute lymphocytic leukemia, glioblastoma, ovarian cancer, breast cancer, colon cancer, bladder cancer, liver cancer and prostate cancer, and increased CD47 expression has been found to be associated with poor prognosis in a variety of malignant tumors.
[0003] There are currently three known natural ligands for CD47: integrin, thrombospondin-1 (TSP-1) and signal-regulatory protein α (SIRPα). CD47 highly expressed in tumor cells can bind to SIRPα on the surface of macrophages, transmit "don't eat me" signals to macrophages, and can overcome the "eat me" signals expressed on tumor cells (such as calreticulin, etc.), thereby shielding the phagocytic function of macrophages and causing tumor immune escape. By blocking the binding of CD47 to SIRPα and cutting off the "don't eat me" signal, the phagocytic ability of macrophages to tumor cells can be restored, and this anti-cancer ability will be widely used against various cancers.
[0004] In addition to blocking the binding of CD47 to SIRPα, CD47 antibody drugs can also assist in killing tumor cells through the following three pathways: ① Promoting DC cells to phagocytose tumor cells, and then presenting antigens to T cells to stimulate anti-tumor adaptive responses; ② Killing tumor cells through NK cell-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement dependent cytotoxicity); ③ Activating tumor cell apoptosis pathways and directly inducing tumor cell apoptosis. According to statistics, there are currently more than 100 drugs targeting CD47 targets in the clinical research stage worldwide, involving monoclonal antibodies, bispecific antibodies, and fusion proteins. Among them, 36 products have entered the clinical stage in China alone, but no drug has been approved for marketing so far.
[0005] The most important issue that prevents CD47 from becoming a drug is that its safety is difficult to ensure. In addition to being highly expressed on the surface of tumor cells, CD47 is also widely expressed on the surface of red blood cells. On the surface of aged red blood cells, CD47 expression is downregulated or undergoes conformational changes, and the "don't eat me" signal is reduced, which promotes the phagocytosis of aged red blood cells by macrophages. It is in this way that CD47 participates in maintaining the balance of red blood cells in the body. This also means that while CD47 targeted drugs kill tumor cells, they will inevitably also accidentally injure red blood cells. In several terminated clinical trials, anemia and thrombocytopenia have also become common side effects, and drugs targeting CD47 will be depleted by red blood cells before binding to tumor cells, greatly reducing the efficacy of the drug.
[0006] Small interfering RNA (siRNA) is an artificially synthesized double-stranded RNA molecule between 15-30 bp, which can target specific sequence mRNA, prevent mRNA translation, and thus achieve the purpose of reducing the expression level of the gene. Since RNA interference therapy targets target gene mRNA, and mature red blood cells do not have genetic materials such as DNA and RNA, RNA interference therapy can effectively avoid red blood cell-related side effects and achieve the purpose of only knocking down the CD47 expression level of tumor cells without affecting red blood cells. Therefore, using siRNA drugs to target and knock down CD47 and block the pairing of CD47 and SIRPα is a very promising new treatment option. Summary of the invention
[0007] The main purpose of the present invention is to provide a siRNA for inhibiting the expression of CD47 gene, so as to solve at least one of the above technical problems.
[0008] According to one aspect of the present invention, siRNA for inhibiting CD47 gene expression is provided.
[0009] The present invention performs sequence design based on the mRNA sequence of human CD47 to obtain a series of siRNAs that can theoretically target and inhibit the expression of human CD47. Through a large number of experimental verifications and screening, five siRNAs that can significantly reduce the expression of CD47 at lower concentrations are finally obtained, which are:
[0010] siRNA-1 (hereinafter referred to as CD47-8), wherein the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 15; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 16;
[0011] siRNA-2 (hereinafter referred to as CD47-18), wherein the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 35; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 36;
[0012] siRNA-3 (hereinafter referred to as CD47-19), wherein the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 37; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 38;
[0013] siRNA-4 (hereinafter referred to as CD47-22), wherein the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 43; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 44;
[0014] The nucleotide sequence of the positive strand of siRNA-5 (hereinafter referred to as CD47-23) includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:45; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:46.
[0015] The siRNA provided by the present invention can knock down the expression level of CD47 by more than 95% at a dosage concentration of 10 nM; and at a dosage concentration of 1 nM, the efficiency of CD47-18, CD47-19, CD47-22 and CD47-23 in inhibiting CD47 gene expression is still maintained at more than 90%, among which CD47-23 can reduce the expression amount of CD47 mRNA by about 98% at a dosage concentration of 1 nM.
[0016] In some embodiments, modification of the sense strand and / or antisense strand may include methoxy modification, methoxyethyl modification or halogen modification of some or all of the nucleotides in the sequence; that is, replacing the hydroxyl group at the 2' position of the sugar ring of some or all of the nucleotides in the sequence with a methoxy group, methoxyethyl group or halogen group.
[0017] In some embodiments, the halogen is selected from at least one of fluorine, chlorine, bromine, and iodine.
[0018] In some embodiments, modifying the sense strand and / or the antisense strand may include replacing a P═O bond between some or all of two adjacent nucleotides in the sequence with a P═S bond.
[0019] In the present invention, unless otherwise specified, in the above and below, capital letters C, G, U, A, and T represent the base composition of nucleotides; lowercase letter "m" represents methoxy modification at the 2' position of the sugar ring of the nucleotide at the position corresponding to the letter "m", that is, the hydroxyl group at the 2' position of the sugar ring of the nucleotide at the position corresponding to the letter "m" is replaced by a methoxy group; lowercase letter "f" represents fluorine modification at the 2' position of the sugar ring of the nucleotide at the position corresponding to the letter "f", that is, the hydroxyl group at the 2' position of the sugar ring of the nucleotide at the position corresponding to the letter "f" is replaced by fluorine; "-" represents that the connecting bond between the two adjacent left and right nucleotides is P=O, and "=" represents that the connecting bond between the two adjacent left and right nucleotides is P=S.
[0020] In some embodiments, the modification method of the sense strand of siRNA is 5'-m=m=mmmmfmfffmmmmmmmm-3'.
[0021] In some embodiments, the modification method of the antisense strand of siRNA is 5'-m=f=mmmfmmmmmmmfmfmmm=m=m-3'.
[0022] In some embodiments, the 5' end, 3' end or other position of the sense strand and / or antisense strand of the siRNA can be covalently linked to cholesterol, N-acetylgalactosamine, fatty acid chains and other ligands for delivery.
[0023] The siRNA provided by the present invention can effectively silence the expression of the target gene CD47, can knock down the expression of CD47 in tumor cells independently without being affected by red blood cells in the human body, has low side effects such as blood toxicity, and can be used to prepare drugs for treating various malignant tumors, chronic inflammation or autoimmune diseases with high expression of CD47.
[0024] In some embodiments, the tumor can be selected from at least one of myeloma, leiomyosarcoma, acute lymphoblastic leukemia, non-Hodgkin's lymphoma, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, glioma, melanoma, lung cancer, liver cancer, non-melanoma skin cancer, renal cell carcinoma, cervical cancer, ovarian cancer, and prostate cancer.
[0025] In some embodiments, the chronic inflammatory or autoimmune disease may be selected from at least one of rheumatoid arthritis, autoimmune encephalitis, multiple sclerosis, autoimmune hepatitis, Crohn's disease, and inflammatory bowel disease.
[0026] In some embodiments, the siRNA provided by the present invention can be delivered by delivery systems such as cationic polymers, nanolipid particles (LNP), cationic liposomes, QTsome, lipopolyplex, microparticles, microspheres, nanoemulsions, etc. Among them, QTsome is a lipid nanoparticle in which quadrivalent (Quaternary) and trivalent (Tertiary) cationic phospholipids coexist. The delivery system has the characteristics of high load and high stability, which can protect siRNA from degradation by enzyme systems in the body and escape from endosomes efficiently after entering cells. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The effect of 28 candidate siRNAs on the expression level of CD47 gene in A549 cells;
[0028] Figure 2 The figure shows the inhibitory effects of different siRNAs at different concentrations on the expression level of CD47 gene in A549 cells. DETAILED DESCRIPTION
[0029] The present invention is further described in detail below in conjunction with the embodiments. The examples are only for explanation and are not intended to limit the present invention in any way. Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; the experimental methods in the examples where specific conditions are not specified are usually carried out according to conventional conditions in the art or according to the conditions recommended by the manufacturer.
[0030] Example 1 siRNA design and synthesis
[0031] Based on the mRNA sequence of human CD47 (Gene ID: 961, NM_001777.4), sequence design was performed to obtain a series of siRNAs that can theoretically target and inhibit human CD47 expression, and 28 of them were screened as the final candidate sequences, whose nucleotide sequences are specifically shown in Table 1.
[0032] All sequences shown in Table 1 were chemically modified, wherein the modification method of the sense chain was m=m=mmmmfmfffmmmmmmmm, and the modification method of the antisense chain was m=f=mmmfmmmmmmfmfmmm=m=m, "m" represents methoxy modification at the 2' position of ribose, "f" represents fluorine modification at the 2' position of ribose, "-" represents a phosphodiester bond, and "=" represents a phosphorothioate bond, and the direction is all from 5' end to 3' end.
[0033] The above 28 siRNAs were synthesized for subsequent experiments.
[0034] Table 1 CD47 siRNA candidate sequences
[0035]
[0036]
[0037]
[0038] Experimental Example 1
[0039] Take the frozen human non-small cell lung cancer cell line (A549), place it in a 37°C water bath to thaw, then add 5 mL of F-12K medium (gibco, 21127022) containing 10% FBS (gibco, 100991148), mix well and centrifuge at 1100 rpm for 5 minutes. After centrifugation, discard the supernatant, resuspend it with F-12K medium containing 10% FBS, transfer it to a culture bottle, and continue to culture it in a 37°C / 5% CO2 incubator.
[0040] When A549 cells were growing well and in the logarithmic growth phase, the culture flask was removed, the supernatant was discarded, 2 mL of PBS (gibco, 10010023) was added for rinsing twice, and 2 mL of 0.25% trypsin-EDTA (Solarbio, T1300) was added for digestion for 5 minutes, and then F-12K medium containing 10% FBS was added to terminate the digestion, the cells were evenly blown and centrifuged (1100 rpm, 5 minutes); after centrifugation, the supernatant was discarded, and the cells were resuspended to 2×10 5 1 mL of resuspended cell suspension was added to each well of a 12-well plate. The plate was then placed in a 37°C / 5% CO2 incubator for overnight culture.
[0041] The CD47 siRNA candidate sequences synthesized in Example 1 were mixed with the transfection reagent lipo3000 (Invitrogen, 92008) and added to the A549 cells plated the day before, and placed in a 37°C / 5% CO2 incubator for transfection for 24 hours.
[0042] After transfection, the supernatant was discarded, PBS was added for rinsing twice, and then 800 μL Trizol (invitrogen, 15596018) was added to each well, lysed on ice for 10 minutes, transferred to EP tubes, 200 μL chloroform (Shanghai test, 10006818) was added to each tube, and after vigorous mixing, it was placed on ice for 10 minutes. Centrifuge at 13000rpm for 15 minutes, carefully aspirate the upper clear liquid after centrifugation, transfer to the EP tube of the heart, add an equal volume of isopropanol (Gaojing Chemical, 20220420), precipitate on ice for 20 minutes, centrifuge at 13000rpm for 10 minutes, discard the supernatant after centrifugation, add pre-cooled 75% ethanol to wash twice, centrifuge at 13000rpm for 10 minutes, discard the supernatant, idle once, 13000rpm, 10 minutes, to completely remove the residual 75% ethanol, carefully aspirate the supernatant and open the lid to air dry for 10 minutes. After adding 10 μL DEPC H2O for resuspending, the concentration was measured.
[0043] Take 1ug of each extracted RNA, add 2μL of gDNA wiper (Novozyme, R312-01), add ddH2O to make up to 10μL, put it in the PCR instrument, set the program at 42℃, 2 minutes. After the program is completed, take it out and add 10×RT Mix (2μL), HiScript III Enzyme Mix (2μL), Oligo dT20VN (1μL), Random hexamers (1μL), RNase-free ddH2O (4μL) to each tube according to the first-strand cDNA synthesis kit (Novozyme, R312-01). After mixing well, put it in the PCR instrument and set the program at 37℃, 15 minutes, 85℃, 5 seconds.
[0044] After taking out the sample, prepare the qPCR reaction system, add 2×qPCR Mix (10μL) (Abm, G891), the first-strand cDNA synthesized in the previous step (2μL), PCR primers (GeneCopoeia, HOP127377) (2μL), RNase-free ddH2O (6μL), mix well, put it into the qPCR instrument, and set the program: ① pre-denaturation (10 minutes); ② denaturation (10 seconds), annealing (20 seconds), extension (15 seconds), 40 cycles; ③ melting curve analysis, heating rate is 0.5℃ / 6 seconds; ④ cooling (30 seconds). After the qPCR reaction is completed, the data is processed and plotted according to the Ct value results, such as Figure 1 shown.
[0045] Depend on Figure 1The results show that some of the 28 candidate siRNAs have a significant inhibitory effect on the expression of CD47 mRNA levels and can significantly inhibit the expression of the CD47 gene. At a dosage concentration of 10nM, the five candidate siRNAs CD47-8, CD47-18, CD47-19, CD47-22, and CD47-23 can knock down the expression level of CD47 by more than 95%, significantly reducing its expression.
[0046] After selecting the five candidate sequences CD47-8, CD47-18, CD47-19, CD47-22, and CD47-23, the concentration was lowered to verify the gene silencing efficiency at the cellular level again. The experimental results are as follows: Figure 2 As shown. Figure 2 As shown in the figure, each candidate sequence still exhibited a significant target gene silencing effect. At a dosing concentration of 1 nM, the mRNA level of the target gene CD47 was significantly reduced compared to that of NC. After reducing the dosing concentration to 1 nM, the inhibitory effect of sequence CD47-8 weakened, and the efficiency of the remaining candidate sequences in inhibiting CD47 gene expression remained above 90%. Among them, the silencing efficiency of sequence CD47-23 was optimal at this concentration, which could reduce the expression of CD47 mRNA by about 98%.
[0047] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.
Claims
1. An siRNA for inhibiting CD47 gene expression, characterized in that: The siRNA has a sense strand and an antisense strand, and is selected from at least one of the following siRNAs: siRNA-1, the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 15; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 16; siRNA-2, the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 35; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 36; siRNA-3, the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 37; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO: 38; siRNA-4, the nucleotide sequence of the sense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:43; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:44; For siRNA-5, the nucleotide sequence of the positive strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:45; the nucleotide sequence of the antisense strand includes a modified or unmodified nucleotide sequence as shown in SEQ ID NO:
46.
2. The siRNA according to claim 1, characterized in that The modification includes methoxy modification, methoxyethyl modification or halogen modification of part or all of the nucleotides in the sequence.
3. The siRNA according to claim 2, characterized in that The halogen is selected from at least one of fluorine, chlorine, bromine and iodine.
4. The siRNA according to any one of claims 1 to 3, characterized in that The modification includes replacing a P═O bond between part or all of two adjacent nucleotides in the sequence with a P═S bond.
5. The siRNA according to claim 4, characterized in that The modification method of the positive strand of the siRNA is 5'-m=m=mmmmfmfffmmmmmmmm-3', wherein "m" indicates methoxy modification at the 2' position of the sugar ring of the nucleotide at the corresponding position, "f" indicates fluorine modification at the 2' position of the sugar ring of the nucleotide at the corresponding position, "-" indicates that the connection bond between two adjacent nucleotides is P=O, and "=" indicates that the connection bond between two adjacent nucleotides is P=S.
6. The siRNA according to claim 5, characterized in that The modification method of the antisense strand of the siRNA is 5'-m=f=mmmfmmmmmmmfmfmmm=m=m-3', wherein, "m" indicates that the 2' position of the sugar ring of the nucleotide at the corresponding position is modified by methoxy, "f" indicates that the 2' position of the sugar ring of the nucleotide at the corresponding position is modified by fluorine, "-" indicates that the connection bond between two adjacent nucleotides is P=O, and "=" indicates that the connection bond between two adjacent nucleotides is P=S.
7. Use of the siRNA according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.
8. The use according to claim 7, characterized in that: The tumor is selected from at least one of myeloma, leiomyosarcoma, acute lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer, osteosarcoma, head and neck squamous cell carcinoma, oral cancer, esophageal cancer, gastric cancer, pancreatic cancer, colon cancer, rectal cancer, glioma, melanoma, lung cancer, liver cancer, non-melanoma skin cancer, renal cell carcinoma, cervical cancer, ovarian cancer, and prostate cancer.
9. Use of the siRNA according to any one of claims 1 to 6 in the preparation of a drug for treating chronic inflammation or autoimmune diseases.
10. The use according to claim 9, characterized in that: The chronic inflammatory or autoimmune disease is selected from at least one of rheumatoid arthritis, autoimmune encephalitis, multiple sclerosis, autoimmune hepatitis, Crohn's disease and inflammatory bowel disease.