Small interfering rnas inhibiting programmed cell death ligand 1 (PD-l1) and use thereof
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- LNCTAC CO LTD
- Filing Date
- 2025-12-04
- Publication Date
- 2026-07-02
Smart Images
Figure CN2025140162_02072026_PF_FP_ABST
Abstract
Description
A group of small interfering RNAs that inhibit programmed death receptor ligand 1 (PD-L1) and their applications Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a group of small interfering RNAs that inhibit programmed death receptor ligand 1 (PD-L1) and their applications. Background Technology
[0002] In recent years, tumor immunotherapy has received widespread attention, becoming a novel treatment method following traditional surgery, radiotherapy, chemotherapy, and targeted therapy. Tumor immunotherapy does not directly attack cancer cells but rather fights tumors by activating the body's immune system, thus exhibiting good efficacy and tolerability. As a representative immunotherapy drug, PD-L1 / PD-1 has achieved great success in the treatment of advanced malignant tumors.
[0003] PD-L1 (programmed cell death 1 ligand 1), initially discovered on the surface of tumor cells and named B7-H1, is an overexpressed immunoglobulin. PD-L1 can also be expressed on the surface of antigen-presenting cells (DCs, macrophages, etc.) and vascular endothelial cells under IFN-γ stimulation, and it can bind to PD-1 on the surface of activated T cells. After PD-1 on T cells binds to PD-L1 on tumor cells or APCs, the two tyrosine residues of PD-1, ITIMs and ITSM, are phosphorylated. Protein tyrosine phosphatase SHP-2 (Src homologous phosphatase 2) is recruited and activated, effectively inhibiting T cell activation and even leading to T cell apoptosis, reduced cytokine production, T cell lysis, and induction of antigen tolerance. Simultaneously, it negatively regulates lymphocyte activation, causing the body's immune surveillance function to fail, allowing cells to evade immune cell attacks, resulting in immune escape, and ultimately leading to tumor development and progression. In immunotherapy, PD-1 / PD-L1 drugs bind to PD-1 or PD-L1 respectively, preventing the interaction between PD-1 and PD-L1, restoring the recognition and killing functions of immune cells, and preventing immune escape of tumor cells.
[0004] In recent years, antibody drugs and small molecule drugs targeting PD-L1 / PD-1 have been launched one after another. Studies have found that PD-1 inhibitors and monoclonal antibodies have a higher incidence of adverse reactions compared to PD-L1, which may be related to the fact that PD-L1 targets also retain the function of PD-L2. Overall, there is a lot of competition in antibody drugs and small molecule drugs targeting PD-L1, while the field of nucleic acid drugs is still in the early stages of development.
[0005] Based on this, the present invention is proposed. Summary of the Invention
[0006] This invention first relates to a group of siRNA molecules targeting the mRNA of programmed cell death 1 ligand 1 (PD-L1):
[0007] The siRNA molecule has a positive strand length of 18-22 bases. The siRNA specifically pairs with a specific region of the target gene. The start site of the specific region of the target gene that specifically pairs with the siRNA is located at the following position on the PD-L1 mRNA:
[0008] (1) 5'UTR region: 27, 43, 69;
[0009] (2) Exon 3: 120–188, 213, 364, 427–438, 488, 632–701, 835, 922;
[0010] (3) Exon 4: 508-566, 631-684;
[0011] (4) Exon 7-3 UTR: 1058-1290, 1532-1626, 1696-1772, 1820-2000, 2167-2173, 2214-2401, 2498-2549, 2883-3254, 3391-3614.
[0012] The target gene of the PD-L1 mRNA is numbered ENST00000381577, and its sequence is shown in SEQ ID NO 215.
[0013] Preferably, the siRNA is shown in Table 1 below.
[0014] Table 1:
[0015] SEQ ID NO 215 (PD-L1 mRNA):
[0016] Furthermore, the siRNA molecule is a chemically modified version containing chemical modifications;
[0017] Preferably, the chemical modification of the siRNA molecule is: thiolation of the phosphate ester bond, 2'-O-methyl modification (OME modification), 5-methyl modification of cytosine, or fluorination modification (F modification).
[0018] Furthermore, the present invention also relates to the following applications of the siRNA molecule:
[0019] (1) Prepare a formulation that inhibits the expression of PD-L1 protein; or
[0020] (2) Preparation of drugs or pharmaceutical compositions that inhibit PD-L1-induced immune tolerance and / or immunosuppression; or
[0021] (3) Prepare drugs or drug compositions for treating cancers with high PD-L1 expression.
[0022] The drug or pharmaceutical composition comprises a therapeutically effective amount of the siRNA molecule, and necessary pharmaceutical excipients or delivery carriers.
[0023] Furthermore, the present invention also relates to the following uses of the said siRNA molecule.
[0024] (1) Treatment of cancers with high PD-L1 expression; or
[0025] (2) Inhibit the expression of PD-L1 protein.
[0026] Furthermore, the present invention also relates to a drug or pharmaceutical composition comprising the siRNA molecule, the drug or pharmaceutical composition comprising a therapeutically effective amount of the siRNA molecule, and necessary pharmaceutical excipients; the drug is used to treat diseases caused by PD-L1 overexpression; preferably, the disease is a tumor.
[0027] The drug or drug composition is an aqueous solvent or an injection; the drug or drug composition is administered via local, intravenous, intramuscular, subcutaneous, or intradermal routes. Attached Figure Description
[0028] Figure 1 shows the effect of some of the siRNAs described in Table 1 on inhibiting PD-L1 protein expression in human breast cancer MDA-MB-231 cells. Detailed Implementation
[0029] Example 1: Design, synthesis and modification of siRNA targeting PD-L1
[0030] PD-L1 mRNA (ENST00000381577) from the Ensembl database was selected as the target gene, and 107 small interfering RNAs (siRNAs, 107 on the sense strand and 107 on the antisense strand) were designed. These siRNA molecules are shown in Table 1 below.
[0031] Table 1. Detailed information about siRNA molecules
[0032] The positions shown in Table 1 are determined by the corresponding positions in the Ensemble database sequence.
[0033] Example 2: Testing the activity of small interfering nucleic acid (siRNA) in an in vitro cell model (A549 human lung cancer cells).
[0034] In this embodiment, the inhibitory effect of the siRNA molecules listed in Table 1 on PD-L1 expression in A549 cells was verified. The specific experimental procedure is as follows:
[0035] (1) Preparation of suspension transfection reagent: Dissolve siRNA dry powder in DEPC water to a concentration of 10 μM. Dilute 10 mM siRNA stock solution to the required concentration using serum-depleted medium (Basalmedia, L530KJ). Dilute Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) with serum-depleted medium. Mix the transfection reagent dilution and siRNA dilution separately to prepare siRNA transfection complexes of the preset concentration or concentration gradient. Mix by pipetting and aspirating 3-5 times and let stand at room temperature for 20 min.
[0036] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 5 × 10⁶ cells / mL. 3 Cells were seeded into 96-well plates, and 100 μl of DMEM medium containing 10% FBS was added to each well. Before transfection, the confluence of A549 cells should be >70% under a microscope. The old medium was discarded and replaced with 50 μl of DMEM medium containing 10% FBS. The siRNA transfection complex prepared in step (1) was added to the 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 5 h, the medium was changed to fresh medium.
[0037] (3) 24 h after transfection, total RNA was extracted from the cells, and the expression of PD-L1 mRNA in the cells was detected by quantitative real-time PCR. The PCR primers and probes used to amplify the internal reference gene Actin and the target gene PD-L1 are shown in Table 2.
[0038] Table 2 PCR primer and probe sequences
[0039] Relative gene expression was calculated using the 2^-ΔΔCT method (Livak method), and the inhibition rate of PD-L1 mRNA expression level was calculated according to the following equation:
[0040] Inhibition rate = (1 - 2^-ΔΔCT) × 100%.
[0041] The experimental groups are as follows:
[0042] Cells treated with siRNAs as indicated by their respective numbers;
[0043] Blank, the blank control group, consists of cells that have not been treated with any siRNA.
[0044] The efficiency of some of the siRNA sequences described in Table 1 in inhibiting PD-L1 mRNA in A549 cells is shown in Table 3 below.
[0045] The results show that the siRNA sequences shown in Table 1 have a significant inhibitory effect on the transcription of PD-L1 mRNA.
[0046] Table 3. Inhibition rate (%) of PD-L1 mRNA in A549 cells after siRNA treatment.
[0047] Example 3: Testing the activity of small interfering nucleic acid (siRNA) in an in vitro cell model (MDA-MB-231 human breast cancer cells).
[0048] In this embodiment, the inhibitory effect of the siRNA molecules listed in Table 1 on PD-L1 expression in MDA-MB-231 cells was verified. The specific experimental procedure is as follows:
[0049] (1) Preparation of suspension transfection reagent: Dissolve siRNA dry powder in DEPC water to a concentration of 10 μM. Dilute 10 M m siRNA stock solution to the required concentration using serum-depleted medium (Basalmedia, L530 KJ). Dilute Lipofectamine 2000 transfection reagent (Invitrogen, 11668-019) or Lipofectamine RNAiMAX transfection reagent (Invitrogen, 13778-150) with serum-depleted medium. Mix the transfection reagent dilution and siRNA dilution separately to prepare siRNA transfection complexes of the preset concentration or concentration gradient. Mix by pipetting and aspirating 3-5 times and incubate at room temperature for 20 min.
[0050] (2) Cell treatment: Cells were plated one day before transfection at a concentration of 5 × 10⁶ cells / mL. 3 Cells were seeded into 96-well plates, and 100 μl of DMEM medium containing 10% FBS was added to each well. Before transfection, the confluence of MDA-MB-231 cells should be >70% under a microscope. The old medium was discarded and replaced with 50 μl of DMEM medium containing 10% FBS. The siRNA transfection complex prepared in step (1) was added to the 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 5 hours, the medium was changed to fresh medium.
[0051] (3) 24 h after transfection, total RNA was extracted from the cells, and the expression of PD-L1 mRNA in the cells was detected by quantitative real-time PCR. The PCR primers and probes used to amplify the internal reference gene Actin and the target gene PD-L1 are shown in Table 2, and the calculation method is the same as in Example 2.
[0052] The efficiency of some of the siRNA sequences described in Table 1 in inhibiting PD-L1 mRNA in MDA-MB-231 cells is shown in Tables 4-1 and 4-2 below.
[0053] The results show that the siRNA sequences shown in Table 1 have a significant inhibitory effect on the transcription of PD-L1 mRNA.
[0054] Table 4-1 Inhibition rate (%) of PD-L1 mRNA in MDA-MB-231 cells after siRNA treatment
[0055] Table 4-2 Inhibition rate (%) of PD-L1 mRNA in MDA-MB-231 cells after siRNA treatment
[0056] Example 4: Testing siRNA activity in an in vitro cell model (MDA-MB-231 human breast cancer cells)
[0057] In this example, the inhibitory effect of the siRNA molecules shown in Table 1 on PD-L1 protein levels in MDA-MB-231 cells was verified. The transfection reagent was prepared as in Example 2, and the experimental steps are as follows:
[0058] (1) Use 6-well plates and 1×10⁶ cells per well. 5 / hole;
[0059] (2) The concentration of siRNA molecules used to treat cells was 10 nM;
[0060] (3) 72 h after siRNA treatment, total protein was extracted from the treated MDA-MB-231 cells using RIPA lysis buffer (beyotime, P0013B) supplemented with PMSF (phenylmethylsulfonyl fluoride, beyotime, ST505) and protease inhibitor.
[0061] (4) Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. After blocking with 5% skim milk powder, the membrane was incubated overnight at 4°C with PD-L1 antibody (Abcam, ab213524) and GAPDH antibody (Thermo Fisher, HRP-60004). The next day, the membrane was incubated with secondary antibody (CST, 7074S) at room temperature for 1 hour. Protein expression levels were observed using an enhanced chemiluminescence detection system (GE Healthcare).
[0062] The effects of some of the siRNAs described in Table 1 on inhibiting PD-L1 protein expression in human breast cancer MDA-MB-231 cells are shown in Figure 1 and Table 5.
[0063] The results show that the siRNA sequences shown in Table 1 have a significant inhibitory effect on the expression of PD-L1 protein.
[0064] Table 5. Inhibition rate of siRNA on PD-L1 protein in MDA-MB-231 cells (%)
[0065] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.
Claims
1. A set of siRNA molecules whose target gene is programmed cell death 1 ligand 1 (PD-L1) mRNA, characterized in that, The siRNA molecule has a positive strand length of 18-22 bases. The siRNA specifically pairs with a specific region of the target gene. The start site of the specific region of the target gene that specifically pairs with the siRNA is located at the following position on the PD-L1 mRNA: (1) 5'UTR region: 27, 43, 69; (2) Exon 3: 120–188, 213, 364, 427–438, 488, 632–701, 835, 922; (3) Exon 4: 508-566, 631-684; (4) Exon 7-3 UTR: 1058-1290, 1532-1626, 1696-1772, 1820-2000, 2167-2173, 2214-2401, 2498-2549, 2883-3254, 3391-3614; The sequence of the PD-L1 mRNA is shown in SEQ ID NO 215.
2. The siRNA molecule according to claim 1, characterized in that, The sense strand sequences of the siRNA molecules are shown in SEQ ID NO 1-107, respectively; The antisense strand sequences of the siRNA molecules are shown in SEQ ID NO 108-214, respectively.
3. The siRNA molecule according to claim 1 or 2, characterized in that, The siRNA molecule is a chemically modified version containing chemical modifications; preferably, the chemical modifications are: thiolation of the phosphate ester bond, 2'-O-methyl modification (OME modification), 5-methyl modification of cytosine, or fluorination modification.
4. The following applications of the siRNA molecule according to any one of claims 1-3: (1) Prepare a formulation that inhibits the expression of PD-L1 protein; or (2) Preparation of drugs or pharmaceutical compositions that inhibit PD-L1-induced immune tolerance and / or immunosuppression; or (3) Prepare drugs or drug compositions for treating cancers with high PD-L1 expression; The drug or pharmaceutical composition comprises a therapeutically effective amount of the siRNA molecule, and necessary pharmaceutical excipients or delivery carriers.
5. A drug or pharmaceutical composition comprising the siRNA molecule according to any one of claims 1-3, characterized in that, The drug or pharmaceutical composition contains a therapeutically effective amount of the siRNA molecule, as well as necessary pharmaceutical excipients; The drug is used to treat diseases caused by high expression of PD-L1; preferably, the disease is a tumor.
6. The drug or drug composition according to claim 5, characterized in that, The drug or drug composition is an aqueous solvent or an injection; the drug or drug composition is administered via local, intravenous, intramuscular, subcutaneous, or intradermal routes.