P-glycoprotein (ABCB1) siRNA and uses thereof

Abstract

The invention relates to ABCB1 siRNA and application thereof, the ABCB1 siRNA can effectively knock down the expression level of ABCB1, and an effective treatment strategy can be provided for overcoming MDR.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to ABCB1 siRNA targeting the human ABCB1 (p-glycoprotein, p-gp) gene and its uses. Background Technology

[0002] Multidrug resistance (MDR) in malignant tumors is one of the major threats faced by many chemotherapy drugs. Overexpression of the ATP-binding cassette (ABC) transporter is associated with MDR. ABCB1 (MDR1) / P-glycoprotein (P-gp), a member of the ABC transporter family, can expel drugs from cells by hydrolyzing ATP, reducing drug accumulation in multidrug-resistant cells and thus leading to resistance. Furthermore, P-glycoprotein also acts as a transporter in the blood-brain barrier, restricting drug permeability from the serous side to these organs. Therefore, inhibiting ABCB1 is an effective therapeutic strategy to overcome MDR. Summary of the Invention

[0003] In order to address the problems existing in the prior art, the purpose of this disclosure is to provide an ABCB1siRNA and its uses.

[0004] In a first aspect, the present invention provides an ABCB1 siRNA, wherein the ABCB1 siRNA comprises a core sequence selected from a combination of the following sense and antisense strands:

[0005] (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.2;

[0006] (2) The sense strand contains the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.4;

[0007] (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.6;

[0008] (4) The sense strand contains the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.8;

[0009] Or a combination of nucleotide sequences that have at least 90% similarity to the above sequences.

[0010] In a second aspect, the present invention provides an RNA delivery system comprising ABCB1 siRNA according to the first aspect of the present invention, and one of the following delivery systems: a lipid-based delivery system (such as micelles, liposomes, and lipid nanoparticles (LNPs)), a polymer-based RNA delivery system, or a nanoparticle-based RNA delivery system.

[0011] In a third aspect, this disclosure provides the use of the ABCB1 siRNA described in the first aspect or the RNA delivery system described in the second aspect in the preparation of a medicament for overcoming multidrug resistance.

[0012] The present invention has at least the following beneficial effects:

[0013] The ABCB1 siRNA provided by this invention can effectively knock down the expression level of ABCB1, and is expected to provide an effective treatment strategy for overcoming MDR. Attached Figure Description

[0014] Figure 1 The results are from a multi-concentration siRNA screening experiment.

[0015] Figure 2 The results are for off-target effect detection.

[0016] Figure 3 For IC 50 Test results. Detailed Implementation

[0017] The present invention will be described in detail below. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Before a detailed description of the invention, the following definitions are provided to better understand it.

[0019] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of...". The expressions "comprising," "including," or "basically / mainly composed of..." are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. Additionally, in this document, the expressions "comprising," "including," or "basically / mainly composed of..." can also be understood as closed-ended expressions in certain circumstances, indicating that they include only the elements, components, parts, or method steps specifically listed after the expression, and exclude any other elements, components, parts, or method steps. Furthermore, in the context of this invention, many embodiments use the expression "composed of...", which should be understood as a closed-ended expression, indicating that it includes only the elements, components, parts, or method steps specifically listed after the expression, and excludes any other elements, components, parts, or method steps.

[0020] Small interfering RNA (siRNA), sometimes called short interfering RNA or silencing RNA, is a double-stranded RNA typically 20 to 25 nucleotides in length. It serves as a tool for studying single-gene function in vivo and in vitro, and represents an attractive new class of therapies, particularly targeting untreatable targets for cancer and other diseases. The double-stranded siRNA molecule is assembled in the cytoplasm into an RNA-induced silencing complex (RISC). One strand of the siRNA (the sense strand) is then degraded, while the other strand (the antisense strand, or guide strand) directs the RISC to recognize and bind to the corresponding site on the mRNA with complementary bases. This allows ribonuclease II within the complex to cleave the target mRNA, thereby regulating the expression of the target gene.

[0021] Previous research by the inventors' team showed that using a DNA nanocarrier to deliver human ABCB1 siRNA can effectively inhibit the growth of doxorubicin-resistant human breast cancer cell line MCF-7R in mice (see Wang Zhaoran, Song Linlin, Liu Qing et al. A Tubular DNA Nanodevice as a siRNA / Chemo-Drug Co-delivery Vehicle for Combined Cancer Therapy.[J].Angew Chem Int Ed Engl,2021,60:2594-2598), further demonstrating that inhibiting ABCB1 is an effective therapeutic strategy to overcome MDR. Providing ABCB1 siRNA with better inhibitory effects will help in the drug development of MDR siRNA.

[0022] Therefore, in a first aspect, the present invention provides an ABCB1 siRNA, wherein the ABCB1 siRNA comprises a core sequence selected from a combination of the following sense and antisense strands:

[0023] (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.2;

[0024] (2) The sense strand contains the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.4;

[0025] (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.6;

[0026] (4) The sense strand contains the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.8;

[0027] Or a combination of nucleotide sequences that have at least 90% similarity to the above sequences.

[0028] As used herein, the term "core sequence" refers to a nucleotide sequence essential for the silencing of the target gene by the siRNA of this invention.

[0029] In some embodiments, the siRNA of the present invention comprises a combination of nucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity to the above sequence, wherein the siRNA maintains the repressive effect on the target gene.

[0030] In some embodiments, the siRNA of the present invention comprises a combination of nucleotide sequences that differ from the above sequence by no more than 3 nucleotides.

[0031] In some embodiments, the siRNA of the present invention is at least 19, 20, or 21 nucleotides in length. Therefore, the core sequence can be 19, 20, or 21 nucleotides in length. The ABCB1 siRNA of the first aspect of the present invention can be 19, 20, 21, 22, or 23 nucleotides in length.

[0032] In addition to the core sequence, the sense or antisense strand of the siRNA of the present invention may also include 1 to 2 3' protrusions of any number of nucleotides; preferably, it includes 2 3' protrusions of any number of nucleotides.

[0033] The term "antisense strand" refers to the strand of siRNA that includes regions that are substantially complementary to the target sequence (e.g., ABCB1 mRNA). If the antisense strand is not perfectly complementary to the target sequence, mismatches can occur in internal or terminal regions of the molecule. Typically, the most tolerant mismatches occur in terminal regions, such as within 5, 4, 3, or 2 nucleotides at the 5' or 3' end of the siRNA.

[0034] In some embodiments, the siRNA of the present invention comprises nucleotide mismatches in the antisense strand. As is known in the art, mismatches at certain positions can significantly increase the function of the siRNA and, in some cases, reduce unwanted antisense strand function. In this document, such mismatches do not result in a significant reduction or loss of function of the siRNA of the present invention.

[0035] In some embodiments, the antisense strand of the siRNA of the present invention includes no more than four mismatches with the target sequence (e.g., ABCB1 mRNA). For example, the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target sequence (e.g., ABCB1 mRNA), and the siRNA maintains the repressive effect of the target gene.

[0036] In some embodiments, the siRNA provided by the present invention exhibits excellent target gene inhibitory activity in in vitro experiments. In some embodiments, the siRNA provided by the present invention exhibits a target gene expression inhibition rate of at least 50%, 60%, 70%, or 80% in in vitro experiments.

[0037] As used herein, unless otherwise specified, “G”, “C”, “A”, “T” and “U” in a nucleotide sequence typically represent nucleotides containing guanine, cytosine, adenine, thymine and uracil as bases, respectively.

[0038] In a preferred embodiment, the ABCB1 siRNA comprises a nucleotide sequence selected from combinations of the following sense and antisense strands:

[0039] (5) The sense strand contains the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.14;

[0040] (6) The sense strand contains the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.16;

[0041] (7) The sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.18;

[0042] (8) The sense strand contains the nucleotide sequence shown in SEQ ID NO.19 and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.20.

[0043] Short hairpin RNA (shRNA) is a short double-stranded RNA structure (19-25 nt) generated based on a stem-loop sequence. It can be introduced into cells using a vector, where it is digested with enzymes to form siRNA, which regulates target genes via RNA interference. Therefore, in one embodiment, a corresponding shRNA can also be designed based on the siRNA described in the first aspect of this invention. The shRNA contains the core sequence of the siRNA described in the first aspect of this invention.

[0044] RNA is relatively unstable and is easily degraded by nucleases in vivo, making it difficult for tissues to absorb, thus limiting its application in vivo.

[0045] On the one hand, the relevant properties of siRNA can be improved through modification, thereby increasing the stability of siRNA, improving the uptake rate of cells, and effectively inhibiting the expression of target genes.

[0046] The nucleotides in the siRNA provided by this invention are each independently modified or unmodified nucleotides. In some embodiments, neither the sense strand nor the antisense strand contains any modification. In some embodiments, the sense strand or the antisense strand contains at least one nucleotide modification. In some preferred embodiments, all nucleotides in the sense strand or the antisense strand are modified nucleotides. These modifications on the nucleotides can be any nucleotide modifications known to those skilled in the art, and these modifications do not cause a significant weakening or loss of the function of the siRNA of this invention in inhibiting ABCB1 gene expression.

[0047] Typically, modifications to siRNA can include phosphate backbone modification, ribose modification, and base modification, such as 5'-terminal cholesterol modification, thiophosphate modification, 2'-methoxy modification, 2'-fluorine modification, 5-methylcytosine modification, 5'-phosphate modification (e.g., vinylphosphonate (VP) modification), deoxy-nucleotide modification, and 3'-terminal deoxy-thymidine (dT) nucleotide modification. Modifications can be performed using various techniques, such as chemical synthesis.

[0048] Therefore, in one embodiment, the modification includes one or more of phosphate backbone modification, ribose modification and base modification.

[0049] In one embodiment, the modification includes one or more of the following: 5'-terminal cholesterol modification, thiophosphate modification, 2'-methoxy modification, 2'-fluoro group modification, 5-methylcytosine modification, 5'-phosphate modification (such as vinylphosphonate (VP) modification), deoxy-nucleotide modification, and 3'-terminal deoxy-thymidine (dT) nucleotide modification.

[0050] In a preferred embodiment, the ABCB1 siRNA comprises a core sequence selected from a combination of the following positive and negative strands:

[0051] (9) The sense strand contains the nucleotide sequence gsgsauGfuGfAfGfuugguuugau (SEQ ID NO.9), and the antisense strand contains the nucleotide sequence asUfscaaAfcCfaacucAfcAfucc (SEQ ID NO.10);

[0052] (10) The sense strand contains the nucleotide sequence gsgsauGfuGfAfGfuugguuugau (SEQ ID NO.11), and the antisense strand contains the nucleotide sequence VPusUfscaaAfcCfaacucAfcAfucc (SEQ ID NO.12);

[0053] Where a, g, c, and u represent 2'-O-methyl A, 2'-O-methyl G, 2'-O-methyl C, and 2'-O-methyl U, respectively; Af, Gf, Cf, and Uf represent 2'-fluoro A, 2'-fluoro G, 2'-fluoro C, and 2'-fluoro U, respectively; s represents a thiophosphate bond; and VP represents vinylphosphonate modification.

[0054] In a preferred embodiment, the ABCB1 siRNA comprises a nucleotide sequence selected from combinations of the following sense and antisense strands:

[0055] (11) The sense strand contains the nucleotide sequence gsgsauGfuGfAfGfuugguuugauga (SEQ ID NO.21), and the antisense strand contains the nucleotide sequence asUfscaaAfcCfaacucAfcAfuccsusg (SEQ ID NO.22);

[0056] (12) The sense strand contains the nucleotide sequence gsgsauGfuGfAfGfuugguuugauga (SEQ ID NO. 23), and the antisense strand contains the nucleotide sequence VPusUfscaaAfcCfaacucAfcAfuccsusg (SEQ ID NO. 24);

[0057] Where a, g, c, and u represent 2'-O-methyl A, 2'-O-methyl G, 2'-O-methyl C, and 2'-O-methyl U, respectively; Af, Gf, Cf, and Uf represent 2'-fluoro A, 2'-fluoro G, 2'-fluoro C, and 2'-fluoro U, respectively; s represents a thiophosphate bond; and VP represents vinylphosphonate modification.

[0058] On the other hand, the properties of siRNA can be further improved by forming ligand-RNA conjugates with suitable ligands to reduce circulation clearance, enhance targeted accumulation and cellular uptake, thereby modulating its pharmacokinetics and pharmacodynamics. These ligands include small molecules, lipids, peptides, antibodies, proteins, carbohydrates, and non-coding RNA (ncRNA).

[0059] Therefore, in one embodiment, the ABCB1 siRNA further includes a ligand; preferably, the ligand is N-acetylglucosamine (GalNAc).

[0060] Efficient RNA delivery can typically be achieved through a variety of RNA delivery systems.

[0061] In a second aspect, the present invention provides an RNA delivery system comprising ABCB1 siRNA according to the first aspect of the present invention, and one of the following delivery systems: a lipid-based delivery system (such as micelles, liposomes, and lipid nanoparticles (LNPs)), a polymer-based RNA delivery system or a nanoparticle-based RNA delivery system, or a nucleic acid-based nanoparticle or nanostructure-based RNA delivery system.

[0062] In a third aspect, this disclosure provides the use of the ABCB1 siRNA described in the first aspect or the RNA delivery system described in the second aspect in the preparation of a medicament for overcoming multidrug resistance.

[0063] Example

[0064] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0065] I. Research Objectives

[0066] 1. Using the doxorubicin-resistant human hepatocellular carcinoma cell line HepG2 / ADR, screened by CRO, to screen 15 pairs of siRNA sequences (naked sequences) to knock down the expression of the human ABCB1 (p-gp) gene.

[0067] 2. Off-target effects of some candidate sequences were detected using the inventor's self-screened human breast cancer cell line, doxorubicin-resistant MCF-7 / ADR cells.

[0068] 3. Detection of IC50 of candidate siRNA modification sequences using the doxorubicin-resistant human breast cancer cell line MCF-7 / ADR cells. 50 .

[0069] II. Experimental Design

[0070] Based on the human ABCB1 (p-gp) gene (NCBI Gene ID: 5243), 15 pairs of siRNA sequences were designed and synthesized, as shown in Table 1 below. The positive control (PC) siRNA used was the human ABCB1 siRNA described in the literature Wang Zhaoran, Song Linlin, Liu Qing et al. A Tubular DNA Nanodevice as a siRNA / Chemo-Drug Co-delivery Vehicle for Combined Cancer Therapy. [J]. Angew Chem Int Ed Engl, 2021, 60: 2594-2598, with the following specific sequence:

[0071] Chain of Justice (5' to 3'): CGGAAGGCCUAAUGCCGAA (SEQ ID NO. 47);

[0072] Antisense chain (5' to 3'): UUCGGCAUUAGGCCUUCCG (SEQ ID NO.48).

[0073] The expression level of p-gp in doxorubicin-resistant cell lines was detected by flow cytometry. Cell lines with high p-gp expression were screened for siRNA to obtain the doxorubicin-resistant human liver cancer cell line HepG2 / ADR and the doxorubicin-resistant human breast cancer cell line MCF-7 / ADR, which have high p-gp expression.

[0074] During screening, the knockdown effects of 15 pairs of naked siRNA sequences were detected using the doxorubicin-resistant human hepatocellular carcinoma cell line HepG2 / ADR. The inhibitory effects of siRNA at three concentrations (1 nM, 10 nM, and 30 nM) were examined, and sequence homology in mice was compared. Candidate sequences with the most significant effects were selected for off-target analysis and IC50 assay. 50 Testing.

[0075] Off-target effects were detected using the doxorubicin-resistant human breast cancer cell line MCF7-ADR. siRNAs with low off-target effects, namely DN02001, DN02013, and DN02020, were selected. DN02020 was further modified (DN02052 and DN02054, specific sequences are shown in Table 1), and then IC50 was measured. 50 Finally, candidate sequences were obtained.

[0076] Table 1: siRNA sequences

[0077]

[0078]

[0079] III. Experimental Materials

[0080] 3.1 Experimental Apparatus

[0081] The experimental instruments used are shown in Table 2.

[0082] Table 2: Experimental Instruments

[0083]

[0084] 3.2 Experimental Reagents

[0085] The specific experimental reagents used are shown in Table 3.

[0086] Table 3: Experimental Reagents

[0087]

[0088]

[0089] IV. Experimental Methods

[0090] 1. Cell Plating: Discard the DMEM medium. Digest the doxorubicin-resistant human hepatocellular carcinoma line HepG2 / ADR with 0.25% trypsin, then terminate the digestion with double the volume of medium. Resuspend the cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in fresh DMEM basal medium, and count the cells. Dilute the cells and mix thoroughly. Plate the cells in 12-well plates with 1 mL of cell suspension per well. After culturing for 24 h, perform transfection.

[0091] 2. Change the culture medium: Before performing the transfection operation, aspirate the DMEM basal medium in the cell culture plate and replace it with Opti-MEM, adding 1.0 mL / well.

[0092] 3. Preparation of siRNA working solutions: The final test concentrations of siRNA used in this example are 30 nM, 10 nM, and 1 nM. Therefore, Opti-MEM was used to dilute the 20.0 μM siRNA stock solution to prepare working solutions with different concentrations (0.36 μM, 0.12 μM, and 0.012 μM) for testing. (During sample loading, the working solution needs to be further mixed with RNAiMAX solution at a 1:1 ratio to prepare a transfection mixture. Then, 200 μL / well of the transfection mixture is added to the cell culture plate containing 1 mL of Opti-MEM in step 2. Therefore, the concentration of the working solution should be 12 times the corresponding final test concentration.)

[0093] 4. Prepare RNAiMAX solution: Mix RNAiMAX and Opti-MEM at a ratio of 3:97 to prepare RNAiMAX solution. Gently mix and let stand at room temperature for about 5 minutes. Each well requires 3 μL RNAiMAX + 97 μL Opti-MEM (i.e., 100 μL RNAiMAX solution per well). The required amount of RNAiMAX and the volume of Opti-MEM need to be calculated according to the amount of experimental sample.

[0094] 5. Prepare transfection mixture: Gently mix 100 μL RNAiMAX solution and 100 μL siRNA working solution at a 1:1 ratio, let stand at room temperature for 10 min, and then loop-drop 200 μL of transfection mixture per well into a 12-well plate.

[0095] 6. Incubate the cell culture plate at 37°C and 5% CO2 for 4–6 hours. Then, add 1 mL of Opti-MEM medium containing 20% ​​FBS to each well and continue culturing at 37°C and 5% CO2 for 48 hours before collecting the cells.

[0096] 7. Remove the culture medium from each well of cells, extract cellular RNA using the TransZolUp Plus RNA Kit, and then measure the RNA concentration using a nucleic acid protein analyzer (where 260 / 280>1.8 and 260 / 230≈2.0 indicate that the extracted nucleic acid has high purity).

[0097] 8. Reverse transcription

[0098] Take 1000ng of RNA and use GoScript. TM Reverse transcription was performed using Reverse TranscriptionMix, Oligo(dT) (reaction system: 20 μL). For samples with a total RNA concentration of less than 1000 ng, the total amount can be adjusted to an appropriate level based on the actual measured RNA concentration.

[0099] Add the following reagents as shown in Table 4 to prepare the GoScript. TM Reverse transcription mixture. For multiple sample detection, the following reagents can be prepared by mixing according to the required number of samples, and then added to each sample in 10 μL / tube.

[0100] Table 4

[0101]

[0102] As shown in Table 5 below, add 10 μL of GoScript TM Mix the reverse transcription mixture with up to 10 μL of RNA sample and place it in a PCR tube.

[0103] Table 5

[0104]

[0105] After mixing thoroughly, proceed with the reaction according to the procedure shown in Table 6. After the reaction is complete, add a certain amount of nuclease-free water to each reaction tube to ensure that the RNA concentration: total volume = 10 ng / μL.

[0106] Table 6

[0107]

[0108] 9. qPCR reaction

[0109] qPCR reactions were performed using Fast Start Universal SYBR Green.

[0110] Prepare a 25 μL qPCR reaction system as shown in Table 7.

[0111] Table 7

[0112]

[0113] Add 25 μL of the above reaction mixture to each well of a 96-well PCR plate, cap the plate, and briefly centrifuge to allow the solution to settle at the bottom. Perform the reaction on a real-time quantitative PCR instrument according to the reaction procedure shown in Table 8.

[0114] Table 8

[0115]

[0116] 10. qPCR Data Processing

[0117] Using relative quantitative 2 -△△Ct The quantitative fluorescence data were analyzed to calculate the relative expression levels of the target genes. ΔΔCt = (Ct target gene in experimental group - Ct internal reference in experimental group) - (Ct target gene in blank control group - Ct internal reference in blank control group) mean.

[0118] Relative expression level (%) = 2 -△△Ct ×100.

[0119] Inhibition rate (%) = (blank control group 2) -△△Ct Mean – Experimental Group 2 -△△Ct ) / Blank control group 2 -△△Ct Mean × 100.

[0120] 11. Off-target effect detection

[0121] Off-target effects were detected using the primers shown in Table 9.

[0122] Table 9

[0123]

[0124]

[0125] Specifically, off-target effects were detected using the following experimental method:

[0126] 11.1 Cell Culture

[0127] The doxorubicin-resistant human breast cancer cell line MCF-7 / ADR was cultured in RPMI 1640 medium containing 10% fetal bovine serum. Cells in the exponential growth phase were collected and resuspended to a cell concentration of 40,000 cells / mL for plating, with 1 mL seeded into each well of a 12-well plate.

[0128] 11.2 Transfection

[0129] Observe the cells under a microscope. Transfection begins when the cell confluence reaches 50-70%. Prepare EP tubes according to the amount of siRNA to be transfected and label them. Add 50 μL of OPTI-MEM and the corresponding volume of siRNA to each tube (the working concentration of siRNA is 10 nM for off-target effect detection experiments, and the transfection time is 24 hours) to prepare mixing solution A. Vortex to mix thoroughly and set aside. Add 3 μL of Lipofectamine to each well. TM Calculate the amounts of 3000 transfection reagent and 50 μL OPTI-MEM and prepare mixed solution B. Vortex the solution to mix thoroughly. Add 50 μL of solution B to each tube of OPTI-MEM and siRNA mixed solution A, vortex thoroughly, and incubate at room temperature for 15 min. Add 100 μL of siRNA-lipid complex (solution A-B mixture) to each well of cells, mix thoroughly using the cross-hatching method, and incubate in a cell culture incubator set at 37°C and 5% CO2.

[0130] The negative control (NC) consisted of 100 μL of lipid complex without siRNA, while the blank control consisted of neither lipid complex nor siRNA.

[0131] 11.3 RNA Extraction and Concentration Determination

[0132] Aspirate the culture medium from the 12-well plate. Add 400 μL of RNA-easy Isolation Reagent to each well, ensuring it fully covers the cell surface. Then, use a pipette to thoroughly lyse the cells. Transfer the lysis buffer to a centrifuge tube, add 160 μL of RNAase-free ddH2O, invert to mix, and incubate at room temperature for 5 min. Centrifuge at 12,500 rpm for 15 min at room temperature.

[0133] Remove the centrifuge tube and carefully aspirate 500 μL of the upper aqueous phase into a new centrifuge tube. Add an equal volume of isopropanol, invert to mix, and incubate at room temperature for 10 min. Centrifuge at 12,500 rpm for 10 min at room temperature and discard the supernatant. Then add 0.5 mL of 75% ethanol to gently wash the RNA precipitate, and centrifuge at 9,100 rpm for 3 min at room temperature, discarding the supernatant. Continue centrifuging at 9,100 rpm for 1 min at room temperature, and remove any remaining ethanol using a micropipette tip.

[0134] Air dry the residual ethanol at room temperature for 2 minutes, then add 20 μL of nuclease-free ddH2O to dissolve.

[0135] 11.4 Quantitative Real-Time PCR (qRT-PCR) Experiment

[0136] Prepare the PCR reaction system according to Table 10 below:

[0137] Table 10

[0138]

[0139] In a 1.5 mL centrifuge tube, add all components except primers and template, labeling it Solution A. Label 1.5 mL EP tubes, add 28 μL of Solution A and 175 ng of total RNA to each of the seven tubes, and mix well. Mix the internal control gene hsACTB, the target gene hsABCB1, and the upstream and downstream primers for off-target effect detection as shown in Table 10, and set aside.

[0140] Add 4 μL of solution B and 1 μL of solution C to each well of the PCR plate according to the arrangement. Cover with sealing film, centrifuge at 2,500 rpm for 1 min, and then transfer to the PCR machine.

[0141] Place the plate in the qRT-PCR instrument and run the following procedure:

[0142] Reverse transcription: 55℃, 15 min;

[0143] Pre-denaturation: 95℃, 30 seconds;

[0144] Cyclic reaction: 95℃, 10 sec; 60℃, 35 sec; 40 cycles;

[0145] Melting curves: 95℃, 15sec; 60℃, 60sec; 95℃, 15sec.

[0146] The running time is approximately 1.5 hours. Analyze the experimental results and calculate 2. -ΔΔCt .

[0147] 12.IC 50 Detection

[0148] IC 50 The experimental procedure for detecting off-target effects is similar to that for detecting off-target effects, except that during cell transfection, at IC50... 50 Multiple siRNA working concentrations were used in the detection experiments, including 10 nM, 2.5 nM, 0.625 nM, 0.156 nM, and 0.039 nM, and the transfection time was 24 hours.

[0149] V. Experimental Results and Data Analysis

[0150] The results of the siRNA multi-concentration screening experiment are shown below Figure 1 As shown in Table 11, the inhibition rate of each pair of siRNAs can be obtained by analyzing the screening results. It can be seen that all tested siRNAs showed comparable or better inhibition rates compared with the positive control (PC) group.

[0151] Table 11: Results of multi-concentration screening of DN02 siRNA

[0152]

[0153] The inventors further compared the homology of siRNA sequences in mice and found that six siRNAs showed high homology with mice, rats, and monkeys. Since subsequent efficacy and toxicology studies required mice, rats, and monkeys, using siRNAs with high homology was more beneficial for experimental design and results analysis. Therefore, siRNAs numbered 1 (DN02001), 4 (DN02004), 13 (DN02013), 15 (DN02015), 19 (DN02019), and 20 (DN02020) were selected to verify off-target effects in human doxorubicin-resistant breast cancer cell line MCF-7 / ADR. The inventors further modified siRNA number 20 (DN02020), which showed the best inhibitory effect, to obtain DN02052 and DN02054. Furthermore, literature indicates that chemical modification of the siRNA seed region can, to some extent, avoid off-target effects (see Aimee L, Jackson, Peter S, Linsley, Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. [J]. Nat Rev Drug Discov, 2010, 9:0). DN02054 and DN02052 differ only in that they have methoxyuridine modification at the 5' end of the antisense strand. Therefore, among DN02020, DN02052, and DN02054, which have essentially the same basic nucleotide sequence, DN02054 was selected for off-target effect verification.

[0154] Candidate sequences with a suppression rate of no more than 50% (fold difference (FC) > 0.5) and higher homology to potential off-target genes were selected by off-target effect detection. Specific experimental results are shown in Table 12 below. Figure 2 As shown. Based on the off-target effect detection results, DN02001 / 013 / 019 / 020(052 / 054) was selected as a candidate sequence.

[0155] Table 12: Results of Off-Target Effect Detection

[0156]

[0157]

[0158] Furthermore, the inventors performed IC50 on the modified siRNA. 50The test results are as follows Figure 3 As shown. IC 50 Experimental results showed that the modified siRNA DN02052(IC 50 =0.09351nM) and DN02054 (IC 50 Both (0.0292 nM) significantly inhibited ABCB1 gene expression in MCF-7R cells, and the 5'-VP modified DN02054 sequence was more effective than the unmodified DN02052 sequence.

[0159] Considering the above results, DN02001 / 013 / 019 / 020(052 / 054) was finally selected as the candidate sequence. Therefore, this invention screened out and modified ABCB1 siRNAs with better ABCB1 inhibitory effects, which will contribute to the development of MDR siRNA drugs.

[0160]

[0161]

Claims

1. An ABCB1 siRNA, wherein, The ABCB1 siRNA contains a core sequence selected from combinations of the following sense and antisense strands: (1) The sense strand contains the nucleotide sequence shown in SEQ ID NO.1, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.2; (2) The sense strand contains the nucleotide sequence shown in SEQ ID NO.3, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.4; (3) The sense strand contains the nucleotide sequence shown in SEQ ID NO.5, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.6; (4) The sense strand contains the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.8; Or a combination of nucleotide sequences that have at least 90% similarity to the above sequences.

2. The ABCB1 siRNA according to claim 1, wherein the sense strand and the antisense strand further comprise 1 to 2 3' protrusions of any number of nucleotides; preferably, it comprises 2 3' protrusions of any number of nucleotides.

3. The ABCB1 siRNA according to claim 1 or 2, wherein the ABCB1 siRNA contains a nucleotide sequence selected from combinations of the following sense and antisense strands: (5) The sense strand contains the nucleotide sequence shown in SEQ ID NO.13, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.14; (6) The sense strand contains the nucleotide sequence shown in SEQ ID NO.15, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.16; (7) The sense strand contains the nucleotide sequence shown in SEQ ID NO.17, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.18; (8) The sense strand contains the nucleotide sequence shown in SEQ ID NO.19 and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.

20.

4. The ABCB1 siRNA according to any one of claims 1-3, wherein the sense strand or the antisense strand comprises at least one nucleotide modification; preferably, all nucleotides of the sense strand or the antisense strand are modified nucleotides.

5. The ABCB1 siRNA according to claim 4, wherein the modification includes one or more of phosphate backbone modification, ribose modification and base modification; preferably, the modification includes one or more of 5'-terminal cholesterol modification, thiophosphate modification, 2'-methoxy modification, 2'-fluorine modification, 5-methylcytosine modification, 5'-phosphate modification (such as vinylphosphonate (VP) modification), deoxy-nucleotide modification, and 3'-terminal deoxy-thymidine (dT) nucleotide modification.

6. The ABCB1 siRNA according to claim 5, wherein the ABCB1 siRNA Contains a core sequence selected from combinations of the following justice chains and antisense chains: (9) The sense strand contains the nucleotide sequence shown in SEQ ID NO.9, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.10; (10) The sense strand contains the nucleotide sequence shown in SEQ ID NO.11 and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.

12.

7. The ABCB1 siRNA according to claim 5, wherein the ABCB1 siRNA Nucleotide sequences comprising combinations of the following sense and antisense strands: (11) The sense strand comprises the nucleotide sequence shown in SEQ ID NO.21, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO.22; (12) The sense strand contains the nucleotide sequence shown in SEQ ID NO.23 and the antisense strand contains the nucleotide sequence shown in SEQ ID NO.

24.

8. The ABCB1 siRNA according to any one of claims 1-7, further comprising a ligand; Preferably, the ligand is GalNAc.

9. An RNA delivery system comprising ABCB1 siRNA according to any one of claims 1-8, and one of the following delivery systems: a lipid-based delivery system (such as micelles, liposomes, and lipid nanoparticles), a polymer-based RNA delivery system, or a nanoparticle-based RNA delivery system, or a nucleic acid-based nanoparticle or nanostructure-based RNA delivery system.

10. Use of the ABCB1 siRNA according to any one of claims 1-8 or the RNA delivery system according to claim 9 in the preparation of a medicament for overcoming multidrug resistance.

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