Silencing siRNA of MMR gene and its combination application

Abstract

This invention belongs to the fields of molecular biology and biomedicine, and discloses a class of siRNAs for silencing MMR genes and their combined applications. This invention provides a class of siRNA sequences capable of inhibiting the expression of key MMR genes MLH1, MSH2, and MSH6. Experimental results show that the siRNAs of this invention can effectively inhibit the expression of target genes. Furthermore, lipid nanocarriers adapted for in vivo delivery of siRNA (such as siMMR@LNPs) are developed based on siRNA. In an MSS CRC mouse model, siMMR@LNPs combined with oxaliplatin significantly inhibited in vivo tumor progression and liver metastasis, activated a strong immune response, and induced the production of tumor neoantigens, which can serve as a novel immunotherapy regimen for MSS CRC.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biomedicine, specifically relating to a class of siRNAs that silence MMR genes and their combined applications. Background Technology

[0002] Colorectal cancer (CRC) is the third leading cause of cancer incidence and the second leading cause of cancer death worldwide. Based on microsatellite instability, CRC can be classified into microsatellite unstable (MSI) and microsatellite stable (MSS) types. MSS CRC accounts for approximately 85% of all CRC cases. It responds poorly to immune checkpoint inhibitors, such as anti-PD-1 / PD-L1 antibodies, and is considered a "cold cancer" type, with clinical needs far from being met.

[0003] The mismatch repair (MMR) system is a key mechanism for maintaining genomic stability, primarily composed of proteins such as MLH1, MSH2, MSH6, and PMS2. Loss of MMR function leads to altered microsatellite sequence lengths (MSI phenotype) and the generation of numerous frameshift mutations and neoantigens. In MSI CRC, this characteristic makes it sensitive to immunotherapy. However, in MSS CRC, MMR function is normal, the mutation burden is low, neoantigens are scarce, and the immune response is weak.

[0004] Oxaliplatin is a third-generation platinum-based anti-tumor drug that plays a central role in the adjuvant and metastatic treatment of colorectal cancer. Its combination with 5-fluorouracil / leucovorin (such as the FOLFOX regimen) has been approved as a standard treatment for colorectal cancer. Oxaliplatin induces tumor cell death by binding to tumor cell DNA to form Pt-DNA adducts, leading to alterations in DNA structure and impaired replication and transcription. Simultaneously, studies have shown that oxaliplatin can significantly increase the gene mutation burden. This property provides a theoretical basis for inducing a highly mutated state in tumors through oxaliplatin.

[0005] However, conventional systemic administration of oxaliplatin is highly toxic, and chemotherapy alone is insufficient to fully activate the anti-tumor immune response, failing to fully translate its mutation-inducing properties into synergistic effects of immunotherapy. Currently, there is still a lack of effective means in clinical practice to specifically regulate MMR function in MSS colorectal cancer, stabilize and induce the MSI phenotype, and then combine it with immunotherapy to improve efficacy. How to overcome the resistance of MSS colorectal cancer to immune checkpoint inhibitors is a key issue that urgently needs to be addressed in the current field of colorectal cancer diagnosis and treatment. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. This invention proposes to inhibit the function of the MMR system by targeting the key MMR genes MLH1, MSH2, and MSH6 with siRNA, and then combining this with oxaliplatin. This can further enhance genomic instability and increase the number of tumor neoantigens, potentially transforming immunologically "cold tumors" into "hot tumors," providing a new combined treatment strategy for improving the immunotherapy efficacy of microsatellite stable colorectal cancer.

[0007] The first aspect of the present invention is to provide a siRNA.

[0008] A second aspect of the present invention is to provide a siRNA combination.

[0009] A third aspect of the present invention is to provide a carrier.

[0010] A fourth aspect of the present invention is to provide a pharmaceutical composition.

[0011] The fifth aspect of this invention aims to provide the use of the siRNA of the first aspect of this invention, the siRNA combination of the second aspect of this invention, the carrier of the third aspect of this invention, or the pharmaceutical composition of the fourth aspect of this invention in the preparation of a medicament for the prevention and / or treatment of tumors.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides an siRNA comprising a sense strand and an antisense strand, said siRNA including siRNA that inhibits the expression of MLH1, MSH2 and / or MSH6; Wherein, the nucleotide sequence of the sense strand of the siRNA that inhibits MLH1 expression is a1) or a2). a1) Any one of the sequences in SEQ ID NO:1-10, SEQ ID NO:63-72, or SEQ ID NO:123-126; A nucleotide sequence that has at least 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology to either sequence a2) or a1) and has the same function as the sequence shown in a1); The nucleotide sequence of the sense strand of the siRNA that inhibits MLH2 expression is either b1) or b2). b1) Any one of the sequences in SEQ ID NO:11-20, SEQ ID NO:73-82, or SEQ ID NO:127-131; b2) is a nucleotide sequence that has at least 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology with either of the sequences in b1) and has the same function as the sequence shown in b1). The nucleotide sequence of the positive strand of the siRNA that inhibits MSH6 expression is c1) or c2). c1) Any one of the sequences in SEQ ID NO:21-30, SEQ ID NO:83-92, or SEQ ID NO:132-138; Either sequence in c2) or c1) has at least 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology and has the same function as the sequence shown in c1).

[0013] In some embodiments of the present invention, the nucleotide sequence of the antisense strand of the siRNA that inhibits MLH1 expression is a3) or a4). a3) Any one of the sequences in SEQ ID NO:32-41, SEQ ID NO:93-102, or SEQ ID NO:139-142; either sequence a4) or a3) has at least 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology and has the same function as the sequence shown in a3). The nucleotide sequence of the antisense strand of the siRNA that inhibits MLH2 expression is b3) or b4). b3) Any one of the sequences in SEQ ID NO:42-51, SEQ ID NO:103-112, or SEQ ID NO:143-147; either b4) or b3) has at least 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology and is a nucleotide sequence with the same function as the sequence shown in b3). The nucleotide sequence of the antisense strand of the siRNA that inhibits MSH6 expression is c3) or c4). c3) Any one of the sequences in SEQ ID NO:52-61, SEQ ID NO:113-122, or SEQ ID NO:148-154; Either sequence in c4) or c3) has at least 60%, 70%, 80%, 90%, 92%, 94%, 96%, 98%, or 99% homology and has the same function as the sequence shown in c3).

[0014] In some embodiments of the present invention, the 3' end of the siRNA has a dangling base consisting of two deoxynucleotides.

[0015] In some embodiments of the present invention, the deoxynucleotide is deoxythymidine nucleotide.

[0016] In some embodiments of the present invention, any site of the siRNA has one or more chemical modifications selected from the following: phosphate thiocyanate (PS) modification, phosphate dithiocyanate (PS2) modification, 2'-O-methyl (2'-OMe) modification, 2'-fluoro (2'-F) modification, 2'-methoxyethyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-C-allyl modification, 2'-deoxy modification, 2'-hydroxyl modification, 5'-morpholine (5'-Mo) modification, locked nucleic acid (LNA) modification, open-ring nucleic acid (UNA) modification, ethylene glycol nucleic acid (GNA) modification, methylphosphonate (MP) modification, methoxypropylmethylphosphonate (MOP) modification, tricyclic DNA (tcDNA) modification, (S)-restricted ethyl bicyclic nucleic acid ((S)-cEt-BNA) modification, indole modification, peptide nucleic acid (PNA) modification, 5'-( E)-vinylphosphonate (VP) modification, N6-methyladenosine (m6A) modification, 5-methylcytidine (m5C) modification, 3-methyluridine (m3U) modification, 5-methyluridine (m5U) modification, pseudouridine modification, 2-thiouridine (s2U) modification, propynouraidine (5-pU) modification, 5'-methylcytosine modification, 5'-ethynyluridine modification, modification by linking the 5' or 3' end of the nucleotide to an inverted abasic nucleotide (invAB), modification by replacing the nucleotide with an inverted abasic nucleotide (invAb), modification by replacing the nucleotide with 2,4-difluorotolyl ribonucleotide (rF) or by replacing the nucleotide with (S)-glycerol nucleic acid, single-chain terminal phosphorylation modification, single-chain terminal cholesterol modification, single-chain terminal galactose modification, single-chain terminal polypeptide modification, single-chain terminal fluorescent probe labeling modification, and ligand modification.

[0017] A second aspect of the present invention provides a siRNA combination comprising at least two siRNAs described in the first aspect of the present invention.

[0018] In some embodiments of the present invention, the siRNA combination includes siRNA that inhibits MLH1 expression, siRNA that inhibits MLH2 expression, and siRNA that inhibits MSH6 expression.

[0019] In some embodiments of the present invention, the proportion of each siRNA in the siRNA combination is greater than 0.1%.

[0020] In some embodiments of the present invention, the proportions of the siRNA that inhibits MLH1 expression, the siRNA that inhibits MLH2 expression, and the siRNA that inhibits MSH6 expression in the siRNA combination are all greater than 0.25%.

[0021] In some embodiments of the present invention, the mass ratio of the siRNA that inhibits MLH1 expression, the siRNA that inhibits MLH2 expression, and the siRNA that inhibits MSH6 expression in the siRNA combination is 1:(1-2):(1-2); preferably 1:1:1.

[0022] A third aspect of the present invention provides a vector loaded with a combination of siRNA from the first aspect of the present invention and siRNA from the second aspect of the present invention.

[0023] In some embodiments of the present invention, the carrier includes at least one of viral carriers, liposomes, lipid nanoparticles, exosomes, GalNAc conjugates, cationic polymers, solid-structure lipid carriers, and protein-based nanocarriers.

[0024] In some embodiments of the present invention, the carrier is lipid nanoparticles.

[0025] In some embodiments of the present invention, the carrier includes ionizable lipid compounds, steroids, neutral lipids, and / or PEG lipids.

[0026] In some embodiments of the present invention, the ionizable lipids include 3-(bisdodecylamino)-N1,N1,4-tridodecyl-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazine diethylamine (KL22), 14,25-bistridecyl-15,18,21,24-tetraaza-octacosane (KL25), and 1,2-dilinolenicooxy-N,N-dimethylaminopropane (DLin-DMA). 2,2-Dilinolenyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) (abbreviated as MC3), 2,2-dilinolenyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-diolenyloxy-N,N-dimethylaminopropane (DODMA), 2-({8- [-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA), (2R)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[- At least one of cholester-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanedioate (L319) and 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}9-heptadecanedioate (SM102).

[0027] In some embodiments of the present invention, the auxiliary lipids include 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-di-undecanoyl-sn-glycerol-3-phosphate choline (DUPC), and 1-palmitoyl-2 oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16LysoPC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docosahexaenoic)-sn-glycerol-3-phosphate choline 3-Phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME16.0PE), 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine), 1,2-di- Sodium oleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), and sphingomyelin; preferably DSPC.

[0028] In some embodiments of the present invention, the steroid includes at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, and brassosterol; preferably cholesterol.

[0029] In some embodiments of the present invention, the PEG lipid comprises at least one of 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (DMG-PEG), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-distearate glycerol (PEG-DSG), PEG-dispalmitoyl, PEG-dioleyl, PEG-distearate, PEG-diacylglycineamide (PEG-DAG), PEG-dispalmitoylphosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA), preferably DMG-PEG, such as DMG-PEG2000.

[0030] In some embodiments of the present invention, the carrier is obtained by including the following steps: The siRNA or siRNA combination was dissolved in sodium acetate solution to obtain an aqueous phase; Ionizable lipid compounds, steroids, neutral lipids, and polymer-bound lipids were dissolved in ethanol and mixed to obtain the alcohol phase. The aqueous phase and the alcohol phase are mixed and reacted to obtain the support.

[0031] In some embodiments of the present invention, the final concentration of the siRNA or siRNA combination in the aqueous phase is 100-150 ng / μL, such as any value or a range formed by any combination of 100, 110, 120, 130, 140 or 150 ng / μL.

[0032] In some embodiments of the present invention, the final concentration of the ionizable lipid compound in the alcohol phase is 2.99-6.5 mg / mL, such as any value or a range formed by any combination of 2.99, 3.5, 4, 4.5, 5, 5.5, 6 or 6.5 mg / mL.

[0033] In some embodiments of the present invention, the final concentration of the steroid in the alcohol phase is 0.93-2.63 mg / mL, such as any value or a range formed by any combination of 0.93, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5 or 2.63 mg / mL.

[0034] In some embodiments of the present invention, the final concentration of the neutral lipid in the alcohol phase is 0.79-3.58 mg / mL, such as any value or a range formed by any combination of 0.79, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2 or 3.58 mg / mL.

[0035] In some embodiments of the present invention, the final concentration of the PEG lipid in the alcohol phase is 0.31-1.09 mg / mL, such as any value or a range formed by any combination of 0.31, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 or 1.09 mg / mL.

[0036] In some embodiments of the present invention, the alcohol phase and the aqueous phase are mixed at a volume ratio of 1:(1-5), such as any ratio of 1:1, 1:2, 1:3, 1:4 or 1:5 or any range of both.

[0037] This invention provides a siRNA that targets and inhibits MLH1, MSH2, and MSH6, and develops a lipid nanocarrier adapted for in vivo delivery of the siRNA to achieve in vitro and in vivo silencing of target gene expression. The LNP drug (siMMR@LNPs) prepared based on the above siRNA, combined with oxaliplatin, significantly inhibits the growth of MSS CRC tumors in vivo, enhances the mutational burden of MSS CRC tumors, induces the production of tumor neoantigens, and activates the anti-MSS CRC T-cell immune response. Simultaneously, siMMR@LNPs combined with oxaliplatin significantly inhibits the growth of MSS CRC liver metastases and reverses tumor-induced liver damage, providing a new strategy for tumor therapy with a complete DNA mismatch repair system.

[0038] A fourth aspect of the present invention provides a pharmaceutical composition comprising siRNA of the first aspect of the present invention, a combination of siRNAs of the second aspect of the present invention, or a carrier of the third aspect of the present invention.

[0039] In some embodiments of the present invention, the pharmaceutical composition further includes at least one DNA mutation inducer.

[0040] In some embodiments of the present invention, the DNA mutation inducer includes at least one of chemical mutagens, physical mutagens, and biological mutagens.

[0041] In some embodiments of the present invention, the chemical mutagen includes alkylating agents (such as diethyl sulfate, diethyl sulfate, nitrogen mustard hydrochloride, etc.), base analogs (such as 6-bromouracil, 6-BudR, maleic hydrazide, 2-aminopurine, etc.), intercalating agents (such as acridine dyes), nitrous acid, sodium azide, hydroxylamine, platinum compounds, antimetabolites (such as folic acid analogs (methotrexate), pyrimidine analogs (such as gemcitabine, capecitabine, 5-fluorouracil, fluorouracil, cytarabine, nitrogen...). The drug contains at least one of the following: uracil and purine analogues and related substances (such as 6-mercaptopurine, 6-thioguanine, pentostatin, etc.); topoisomerase inhibitors (such as camptothecin, 9-aminocamptothecin, topotecan, ixotecan, irinotecan, belotecone, etoposide, anthracyclines (such as doxorubicin, daunorubicin, idarubicin, epirubicin, pirarubicin, pentorubicin, meparone, ICRF-193, dexrazosen, arubicin, neomycin, roscillaridin A, etc.); preferably platinum compounds.

[0042] In some embodiments of the present invention, the platinum compound includes at least one of oxaliplatin, cisplatin, carboplatin, nedaplatin, lobaplatin, picaplatin, ceterplatin, and cyclic platinum; preferably oxaliplatin.

[0043] In some embodiments of the present invention, the biomutant includes a base editor.

[0044] In some embodiments of the present invention, the pharmaceutical composition further includes combination drugs.

[0045] In some embodiments of the present invention, the combined drugs include antitumor drugs.

[0046] In some embodiments of the present invention, the antitumor drug is selected from at least one of chemotherapy drugs, radiotherapy drugs, photosensitizers, photothermal agents, immunotherapy drugs, or drugs that relieve immunosuppression.

[0047] In some embodiments of the present invention, the chemotherapeutic agent is selected from at least one of alkylating agents, platinum-based drugs, podophyllotoxins, camptothecin-based drugs, taxane-based drugs, anthracyclines, fluoropyrimidines, or antibiotics.

[0048] In some embodiments of the present invention, the chemotherapeutic agent comprises (a) alkylating agents, such as nitrogen mustard (e.g., nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimine and methylmelamine (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorurea, streptozotocin), and triazines (e.g., dacarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., gemcitabine, capecitabine, 5-fluorouracil, fluorouracil, cytarabine, aziridine), and purine analogs and related substances (e.g., 6-mercaptopurine). (c) Natural products, such as vinca alkaloids (e.g., vincristine, vinblastine), epipodophyllotoxin (e.g., etoposide, teniposide), antibiotics (e.g., actinomycin D, daunorubicin, doxorubicin, mitomycin, epirubicin, bleomycin, pricamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), biological response modifiers (e.g., interferon-α); (d) Other reagents, such as at least one of platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocortical inhibitors (e.g., paclitaxel and mitotane).

[0049] In some embodiments of the present invention, the chemotherapeutic drugs include at least one of gemcitabine, oxaliplatin, paclitaxel, camptothecin, 5-fluorouracil, cisplatin, doxorubicin, mitomycin, or epirubicin.

[0050] In some embodiments of the present invention, the radiotherapy drug is selected from at least one of the drugs used in external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiotherapy (IORT).

[0051] In some embodiments of the present invention, the photosensitizer is selected from at least one of dipyrrole, dihydroporphyrin, or Bengal red.

[0052] In some embodiments of the present invention, the photothermal agent is selected from at least one of noble metal nanoparticles, organic polymers, carbon-based nanomaterials, magnetic nanomaterials, or semiconductor nanomaterials.

[0053] In some embodiments of the present invention, the immunotherapeutic drug comprises at least one of monoclonal antibodies, immune checkpoint inhibitors, antibodies against inhibitory signaling pathway molecules, inhibitors of inhibitory signaling pathway molecules, antibodies against inhibitory second signaling pathway molecules, inhibitors of inhibitory second signaling pathway molecules, PD-L1 inhibitors, PD-1 / PD-L1 monoclonal antibody drugs, immune cells, oncolytic viruses, and tumor vaccines.

[0054] In some embodiments of the present invention, the target of the monoclonal antibody is selected from one of CTLA-4, PD-1, PD-L1, PD-L2, CD20, HER2, VEGF / VEGFR, EGFR, CD19, FGL1, CD47, CD3, CD30, CD33, CD38, CD52, αVβ3, α5β1, FAP, Tenascin, CEA, EPCAM, PSMA, GAN-GD2, GAN-GD3, GM2, and IGF-IR.

[0055] In some embodiments of the present invention, the immune checkpoint inhibitor is an inhibitor that acts on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands.

[0056] In some embodiments of the present invention, the molecules acting on negative co-stimulatory (co-inhibitory) T cells and / or their respective ligands are selected from CTLA-4 (e.g., ipilimumab, ticilimumab; CP-675, 206, AGEN-1884, ATOR-1015, MGD019 (PD-1 / CTLA-4 bispecific antibody)) and PD-1 (e.g., nivolumab, pembrolizumab). embrolizumab, tremelimumab, tislelizumab (BGB-A317), spartalizumab, MEDI0680, PDR001, FAZ053, MGA012 (retifanlimab), sintilimab, toripalimab, cemiplimab, MGD019 (PD-1 / CTLA-4 bispecific antibody), MGD013 (tebotelimab (PD-1 / LAG-3 bispecific antibody)), PD-L1 inhibitors (e.g., atezolizumab, camrelizumab, durvalumab, avelumab), LY3300054, CX-072 (Proclaim-CX-072) FAZ053, KN035, MDX-1105), PD-L2, B7-1, B7-2, B7-H3 (e.g., enoblituzumab, MGD009, MGC018), B7-H4, B7-H6, A2aR (e.g., CPI-444, PBF509), IDO (e.g., GDC0919 (navoximod), epacadostat, indoximid, BMS) 986205), TIM-3 (e.g., TSR022 (TIM-3 monoclonal antibody), MBG453 (TIM-3 monoclonal antibody)), BTLA, VISTA, TIGIT (e.g., BMS-986207, AB154, COM902 (CGEN-15137), OMP-313M32), LAG-3 (e.g., BMS986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-5250), IMP321, TSR-033, LAG525, BI754111, FS-118, MGD013 (tebotelimab,PD-1 / LAG-3 bispecific antibodies), CD40 (e.g., BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9 or dacetuzumab), CD20 (e.g., rituximab (RITUXAN; IDEC-102; IDEC-C2B8), ABP798, ofatumumab or obinutuzumab), CD96, CD73 (e.g., MEDI9447 (oleclumab), CD160 (e.g., BY55), STING, CEA (e.g., cergutuzumab) amunaleukin (RG7813, RO-6895882 or RG7802 (RO6958688)), CD47 (e.g., HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231 or Effi-DEM), PVRIG antibodies (e.g., COM701 (CGEN-15029)), LAIR1, 2B4, KIR (e.g., liri Lumbab (1-7F9, BMS-986015, IPH2101, IPH4102), CEACAM1 (e.g., CM-24 (MK-6018)), GARP (e.g., ARGX-115), PS, CSF1R (e.g., pexidartinib, LY3022855, FPA008, BLZ945), CD94 / NKG2A, TDO, TNFR, and DCR3 are one or more of these.

[0057] In some embodiments of the present invention, the inhibitors acting on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands comprise any one of (b1)-(b3): (b1) Antibodies that specifically bind (neutralize) negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands to T cells; (b2) Specific binding (neutralization) of ligand proteins or peptides that act on negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands on T cells; (b3) Non-protein compounds that specifically bind (neutralize) to negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands on T cells.

[0058] In some embodiments of the present invention, the immune cells include at least one of chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), T cell receptor chimeric T cells (TCR-T), tumor-infiltrating immune cells (TILs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells.

[0059] In some embodiments of the present invention, the oncolytic virus comprises at least one of the following: alpha virus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus, and parvovirus.

[0060] In some embodiments of the present invention, the tumor vaccine comprises at least one of dendritic cell (DC) vaccines, nucleic acid vaccines, and peptide vaccines.

[0061] In some embodiments of the present invention, the immunosuppressant is selected from at least one of mercaptopurine, azathioprine, methotrexate, cyclosporine A, tacrolimus, ruxolitinib, rovaxitinib, tripterygium glycoside, or mycophenolate mofetil.

[0062] In some embodiments of the present invention, the pharmaceutical composition is provided in liquid or solid form.

[0063] In some embodiments of the present invention, the pharmaceutical composition is used as a medicine. Therefore, depending on the specific dosage form used, it may be any one of the following: powder, granules, tablets, capsules, sustained-release formulations, solutions, syrups, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, chewable tablets, effervescent tablets, ointments, emulsions, and lavage solutions.

[0064] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0065] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, emulsion, suspension, syrup, drops, dry suspension, chewable tablet, and effervescent tablet.

[0066] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage forms include injectable dosage forms (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (e.g., sprays, aerosols, powder inhalers, etc.); skin dosage forms (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0067] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is an oral dosage form or an injection.

[0068] In some embodiments of the present invention, the composition further includes one or more pharmaceutically or food-acceptable excipients.

[0069] In some embodiments of the invention, the pharmaceutically or food-acceptable excipient is selected from pharmaceutically or food-acceptable excipients, diluents, or carriers. Pharmaceutically or food-acceptable excipients, diluents, or carriers are well known in the pharmaceutical or food industries. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, or sorbitol. Examples of suitable diluents include ethanol, glycerol, and water. The choice of drug carrier, excipient, or diluent can be based on the intended route of administration and standard pharmaceutical practice. The composition may contain any suitable binder, lubricant, suspending agent, coating agent, or solubilizer, or other than a carrier, excipient, or diluent. Examples of suitable binders include starch, gelatin, natural sugars, and natural or synthetic gums. Natural sugars include, for example, glucose, anhydrous lactose, free-flowing lactose, β-lactose, or corn sweeteners. Natural or synthetic gums include, for example, gum arabic, astragalus gum, sodium alginate, carboxymethyl cellulose, or polyethylene glycol. Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, or sodium chloride. Preservatives, stabilizers, dyes, and even flavorings may be provided in the composition. Examples of preservatives include sodium benzoate, sorbic acid, or parabens. Antioxidants and suspending agents may also be used.

[0070] A fifth aspect of the invention provides the use of the siRNA of the first aspect of the invention, the siRNA combination of the second aspect of the invention, the carrier of the third aspect of the invention, or the pharmaceutical composition of the fourth aspect of the invention in the preparation of a medicament for the prevention and / or treatment of tumors.

[0071] In some embodiments of the present invention, the tumor comprises a tumor with an intact DNA mismatch repair system.

[0072] In some embodiments of the present invention, the tumor is a cold tumor.

[0073] The term "cold tumor" refers to immunosuppressive tumors, characterized by minimal T-cell infiltration within and around the tumor. Cold tumors are a type of tumor characterized by limited T-cell infiltration within and around the tumor, resulting in limited therapeutic efficacy of immune checkpoint inhibitors.

[0074] In some embodiments of the present invention, the tumor includes at least one of solid tumors, soft tissue tumors, hematopoietic tumors, adenomas, or metastatic tumors.

[0075] In some embodiments of the invention, the hematoma comprises a tumor of at least one of blood, lymph, or bone marrow.

[0076] In some embodiments of the present invention, the tumors include oral cancer, salivary gland cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, nasal cavity cancer, sinus cancer, laryngeal cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, small intestinal cancer, appendix cancer, colon cancer, rectal cancer, colorectal cancer, anal canal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, hilar bile duct cancer, distal bile duct cancer, hepatopancreatic ampulla cancer, pancreatic cancer, gastric neuroendocrine tumor, duodenal and ampulla neuroendocrine tumor, jejunal-ileal neuroendocrine tumor, appendiceal neuroendocrine tumor, colorectal neuroendocrine tumor, pancreatic neuroendocrine tumor, thymic cancer, lung cancer, malignant pleural mesothelioma, angiosarcoma, desmoidoma, Ewing sarcoma, fibrosarcoma, gastrointestinal stromal tumor, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, myxofibrosarcoma, and malignant peripheral nerve sheath tumor. Rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, cutaneous fibrosarcoma protuberans, Merkel cell carcinoma, cutaneous malignant melanoma, vulvar cancer, vaginal cancer, cervical cancer, endometrial cancer, uterine carcinosarcoma, endometrial sarcoma, ovarian cancer, fallopian tube cancer, primary peritoneal cancer, gestational trophoblastic tumor, penile cancer, prostate cancer, testicular cancer, renal cancer, renal pelvis cancer, ureteral cancer, bladder cancer, urethral cancer, eyelid cancer, conjunctival cancer, conjunctival melanoma, uveal melanoma, retinoblastoma, lacrimal gland cancer, orbital sarcoma, lymphoma of the ocular adnexa, brain cancer, spinal cord tumor, differentiated thyroid carcinoma, undifferentiated thyroid carcinoma, medullary thyroid carcinoma, parathyroid carcinoma, adrenocortical carcinoma, Hodgkin lymphoma, non-Hodgkin lymphoma, cutaneous lymphoma, plasma cell myeloma, or leukemia.

[0077] In some embodiments of the present invention, the tumor comprises a tumor caused by bacteria or a virus.

[0078] In some embodiments of the present invention, the bacteria or virus includes at least one of hepatitis B virus, hepatitis C virus, human papillomavirus, human cytomegalovirus, Epstein-Barr virus, Helicobacter pylori, or Fusobacterium nucleatum.

[0079] In some embodiments of the present invention, the drug achieves prevention and / or treatment of tumors through any of the following pathways: (a) Inhibit tumor volume growth; (b) Inhibit tumor weight increase; (c) Inhibit tumor cell growth; (d) Improve tumor treatment response rate; (e) Improve the efficacy of immunosuppressive drugs; (f) Inhibit tumor cell metastasis; (g) Improve the efficacy of chemotherapy drugs; (h) Inhibit tumors by regulating the subject's immune system; (i) Promote or activate the subject's immune system to kill tumors or inhibit tumor growth.

[0080] In some embodiments of the present invention, the prevention and / or treatment of tumors further includes the use of one or more chemotherapeutic drugs, radiotherapy drugs, targeted drugs, photosensitizers, photothermal agents, immunotherapy drugs, and / or immunosuppressive drugs.

[0081] In some embodiments of the present invention, the chemotherapeutic agent comprises (a) alkylating agents, such as nitrogen mustard (e.g., nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimine and methylmelamine (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorurea, streptozotocin), and triazines (e.g., dacarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., gemcitabine, capecitabine, 5-fluorouracil, fluorouracil, cytarabine, aziridine), and purine analogs and related substances (e.g., 6-mercaptopurine). (c) Natural products, such as vinca alkaloids (e.g., vincristine, vinblastine), epipodophyllotoxin (e.g., etoposide, teniposide), antibiotics (e.g., actinomycin D, daunorubicin, doxorubicin, mitomycin, epirubicin, bleomycin, pricamycin, and mitoxantrone), enzymes (e.g., L-asparaginase), biological response modifiers (e.g., interferon-α); (d) Other reagents, such as at least one of platinum coordination complexes (e.g., cisplatin, carboplatin), substituted ureas (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine), and adrenocortical inhibitors (e.g., paclitaxel and mitotane).

[0082] In some embodiments of the present invention, the radiotherapy drug is selected from at least one of the drugs used in external radiotherapy, internal radiotherapy, radioimmunotherapy, or intraoperative radiotherapy (IORT).

[0083] In some embodiments of the present invention, the photosensitizer is selected from at least one of dipyrrole, dihydroporphyrin, or Bengal red.

[0084] In some embodiments of the present invention, the photothermal agent is selected from at least one of noble metal nanoparticles, organic polymers, carbon-based nanomaterials, magnetic nanomaterials, or semiconductor nanomaterials.

[0085] In some embodiments of the present invention, the immunotherapeutic drug comprises at least one of monoclonal antibodies, immune checkpoint inhibitors, immune cells, oncolytic viruses, and tumor vaccines.

[0086] In some embodiments of the present invention, the target of the monoclonal antibody is selected from one of CTLA-4, PD-1, PD-L1, PD-L2, CD20, HER2, VEGF / VEGFR, EGFR, CD19, FGL1, CD47, CD3, CD30, CD33, CD38, CD52, αVβ3, α5β1, FAP, Tenascin, CEA, EPCAM, PSMA, GAN-GD2, GAN-GD3, GM2, and IGF-IR.

[0087] In some embodiments of the present invention, the immune checkpoint inhibitor is an inhibitor that acts on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands.

[0088] In some embodiments of the present invention, the negative co-stimulatory (co-inhibitory) molecules acting on T cells and / or their respective ligands are selected from one or more of CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2aR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, CD20, CD96, CD73, CD160, STING, CEA, CD47, PVRIG, LAIR1, 2B4, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and DcR3.

[0089] The beneficial effects of this invention are: This invention provides a class of siRNA sequences capable of inhibiting the expression of key MMR genes MLH1, MSH2, and MSH6. Experimental results show that the siRNA of this invention can effectively inhibit the expression of target genes. Furthermore, lipid nanocarriers adapted for in vivo delivery of siRNA (siMMR@LNPs) were developed based on the siRNA. In an MSS CRC mouse model, siMMR@LNPs combined with oxaliplatin significantly inhibited in vivo tumor progression and liver metastasis, activated a strong immune response, and induced the production of tumor neoantigens, potentially serving as a novel immunotherapy regimen for MSSCRC. Attached Figure Description

[0090] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a dose-response diagram of MLH1 (SEQ 61), MSH2 (SEQ 65), and MSH6 (SEQ 70) siRNAs provided by this invention in CT26 cells. In the diagram, ns indicates no significant difference. express P <0.05, express P <0.01, express P <0.001.

[0091] Figure 2 This figure shows the interference effect of siMMR provided by the present invention on the expression of MLH1, MSH2, and MSH6 proteins in CT26 cells. In the figure, ns indicates no significant difference. express P <0.001.

[0092] Figure 3 This image shows the interference effect of siMMR provided by the present invention on the expression of MLH1, MSH2, and MSH6 mRNA in HCT116 cells. In the image, express P <0.001.

[0093] Figure 4 This image shows the interference effect of siMMR provided by this invention on the expression of MLH1, MSH2, and MSH6 mRNA in DLD1 cells. In the image, express P <0.01, expressP <0.001.

[0094] Figure 5 The image shows the weight of isolated mouse tumors provided by this invention. In the image, express P <0.05, express P <0.01, express P <0.001.

[0095] Figure 6 The image shows the expression of MLH1, MSH2, and MSH6 proteins in mouse tumors as provided by this invention, with a scale bar of 100 μm. In the image, express P <0.01, express P <0.001.

[0096] Figure 7 A graph showing the number of neoantigens in tumor tissue provided by this invention.

[0097] Figure 8 This is an immunogenicity diagram of the neoantigen peptide provided by the present invention. In the diagram, express P <0.05, express P <0.001.

[0098] Figure 9 The present invention provides an immunohistochemical analysis of CD8 in the tumor microenvironment. + Map showing T cell infiltration and GZMB secretion levels, scale bar at 75 μm. In the figure, express P <0.05, express P <0.001.

[0099] Figure 10 The image shows the luminescence intensity of a mouse liver, as provided by this invention. express P <0.01, express P <0.001.

[0100] Figure 11 Gross images and weight diagrams of mouse livers provided for this invention. In the figures, ns indicates no significant difference. express P <0.05, express P <0.001.

[0101] Figure 12 The image shows a mouse liver stained with hematoxylin and eosin (HE) according to the present invention, with a scale bar of 500 μm.

[0102] Figure 13 The graph shows mouse blood ALT and AST levels provided for this invention. In the graph, ns indicates no significant difference.

[0103] Figure 14 This invention provides an immunohistochemical analysis of the protein expression levels of MLH1, MSH2, and MSH6 in MSS CRC liver metastases, with a scale bar of 100 μm. In the figure, express P <0.001.

[0104] Figure 15 The immunohistochemical analysis of mouse MSS CRC liver metastases provided by this invention utilizes CD8+. + A map showing the level of T cell infiltration, with a scale bar of 100 μm. In the figure, express P <0.001. Detailed Implementation

[0105] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0106] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0107] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0108] Example 1: Design and Screening of MLH1, MSH2, and MSH6 siRNAs The full-length sequences of mouse MLH1 mRNA (NM_026810.2), MSH2 mRNA (NM_008628.3), MSH6 mRNA (NM_010830.2), human MLH1 mRNA (NM_000249.4), MSH2 mRNA (NM_000251.3), and MSH6 mRNA (NM_000179.3) were obtained from the National Center for Biotechnology Information (NCBI) database. Based on siRNA design principles, such as controlling GC content to 35%-55%, avoiding continuous single sequences that reduce the intrinsic stability of double strands, avoiding hairpin structures caused by inverted repeat sequences, and avoiding mRNA action sites with complex secondary structures, multiple pairs of siRNA sequences were designed using online siRNA design software, selecting different sites in the coding sequences (CDS) of the MLH1, MSH2, and MSH6 genes. To further verify the efficacy of siRNAs in animal experiments, including mice, human-mouse homologous siRNAs were designed. Specifically, 10 siRNAs each target the human MLH1, MSH2, and MSH6 genes; 10 siRNAs each target the mouse MLH1, MSH2, and MSH6 genes; and 4 siRNAs targeting both MLH1, MSH2, and MSH6 (specific sequences are shown in Tables 1-3). In addition, dTdT hanging ends were designed to increase the stability of the siRNAs, and a negative control NC siRNA was also designed. All designed siRNAs were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. HCT116 / CT26 cells were purchased from ATCC.

[0109] Table 1. Human MLH1, MSH2, and MSH6 siRNA sequences and silencing efficiencies

[0110] Table 2. Murine MLH1, MSH2, and MSH6 siRNA sequences and silencing efficiencies

[0111] Table 3. Human-mouse co-targeting MLH1, MSH2, and MSH6 siRNA sequences and silencing efficiencies

[0112] The silencing efficiency of each siRNA was further evaluated based on its species preference. Specifically, the silencing efficiency of human siRNA was evaluated in HCT116 cells, the silencing efficiency of mouse siRNA sequences was evaluated in CT26 cells, and the silencing efficiency of human-mouse co-targeting siRNAs was detected in HCT116 cells. The specific experimental steps are as follows: Step 1.1: HCT116 / CT26 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and placed in a cell culture incubator with 5% CO2, 37°C and saturated humidity. The cells were then passaged using 0.25% trypsin digestion.

[0113] Step 1.2: Take HCT116 / CT26 cells from passages 6-10, digest and count them, then divide them into groups of 1×10⁶ cells per well. 5 Cells were seeded at a density of 70%-80% in 12-well plates. After 24 hours, when the cell density reached 70%-80%, siRNA transfection was performed. A blank control group (Ctrl), a negative control group (NC), and each siRNA group were included.

[0114] Step 1.3: Perform transfection according to the Lipofectamine™ 3000 (Lipo3000) transfection reagent operation guide. The final concentration of NC and each siRNA for transfection is 50 nM.

[0115] Step 1.4: After 48 h of transfection incubation, total RNA was extracted from cells according to the instructions of the RNA extraction kit (ES Science, RN001). The obtained RNA was dissolved in 40 µL of RNase-free water, and the RNA content and purity of each group were determined by Nanod Drop. Using 1 µg of total RNA as a template, cDNA was synthesized according to the instructions of the iScript™ cDNA Synthesis Kit (Bio-Rad, 1708891), and quantitative qPCR detection of the target genes MLH1, MSH2, and MSH6 mRNA was performed according to the instructions of the iQTM SYBR® Green Supermix (Vazyme, Q711).

[0116] The specific sequences of the siRNAs designed in this invention and their silencing efficiencies in corresponding cells are shown in Tables 1-3. Based on the silencing efficiencies of different siRNAs, h / mMLH1-1 (SEQ ID NO:123), h / mMSH2-1 (SEQ ID NO:127), and h / mMSH6-1 (SEQ ID NO:132) were finally selected for subsequent experiments.

[0117] Example 2: Dose-response relationship of human-mouse co-targeting MLH1, MSH2, and MSH6 siRNAs in CT26 cells This embodiment examines the dose-response relationship of the human-mouse co-targeting MLH1 (i.e., h / mMLH1-1 as shown in SEQ ID NO:123), MSH2 (i.e., h / mMSH2-1 as shown in SEQ ID NO:127), and MSH6 (i.e., h / mMSH6-1 as shown in SEQ ID NO:132) siRNA designed in Example 1 on CT26 cells. The specific experiments are as follows: Step 2.1: CT26 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and placed in a cell culture incubator with 5% CO2, 37°C and saturated humidity. The cells were then passaged using 0.25% trypsin.

[0118] Step 2.2: Take CT26 cells from passage 6-0, digest and count them, then fill each well with 2.5 × 10⁻⁶ cells. 5 Cells were seeded at a density of 100% in 6-well plates and transfected with siRNA when the cell density reached 70%-80% after 24 hours. A blank control group (Ctrl), a negative control group (NC), and groups with different siRNA concentrations were included.

[0119] Step 2.3: Perform transfection according to the Lipofectamine™ 3000 (Lipo3000) transfection reagent operation guide. The final concentration of si-NC for transfection is 50 nM, and the final concentrations of each siRNA are 0.2, 2, 20, and 200 nM, respectively.

[0120] Step 2.4: After transfection and incubation for 24 h, total cell protein was extracted and the protein concentration of each group was determined by BCA protein quantification kit (Beyotime, P0010). 20 μg of protein from each group was taken for Western blot analysis of the expression levels of MLH1, MSH2, and MSH6 proteins.

[0121] Experimental results are as follows Figure 1 As shown. Figure 1 The dose-response relationship of MLH1, MSH2, and MSH6 siRNAs in CT26 cells was investigated. The results showed that the interference of MLH1, MSH2, and MSH6 siRNAs with target protein expression in CT26 cells was dose-dependent in the range of 0.2-200 nM.

[0122] In subsequent applications, MLH1, MSH2, and MSH6 siRNAs were simultaneously transfected into cells at a mass ratio of 1:1:1. Therefore, the 1:1:1 mixture of MLH1, MSH2, and MSH6 siRNAs (i.e., a mixture of h / mMLH1-1, h / mMSH2-1, and h / mMSH6-1 in a 1:1:1 ratio) is uniformly referred to as siMMR.

[0123] Example 3: The interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 proteins in CT26 cells. This embodiment investigates the interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 proteins in CT26 cells. The specific experimental steps are as follows: Step 3.1: CT26 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and placed in a cell culture incubator with 5% CO2, 37°C and saturated humidity. The cells were then passaged using 0.25% trypsin.

[0124] Step 3.2: Take CT26 cells from passages 8-10, digest and count them, and then divide them into groups of 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 70%-80% in 6-well plates. After 24 hours, when the cell density reached 70%-80%, siRNA transfection was performed. Three groups were set up: a PBS group, an NC siRNA group, and a siMMR group. Each group had three parallel wells for biological replicates.

[0125] Step 3.3: Perform transfection according to the Lipofectamine™ 3000 (Lipo3000) transfection reagent operation guide. The transfection NC siRNA is 150 nM / well, and the final siMMR concentration is 50 nM / siRNA, i.e., 150 nM / well.

[0126] Step 3.4: After transfection and incubation for 24 h, total cell protein was extracted, and the protein concentration of each group was determined by BCA protein quantification kit. 20 μg of protein from each group was taken for Western blot analysis of the expression levels of MLH1, MSH2, and MSH6 proteins.

[0127] Experimental results are as follows Figure 2 As shown. Figure 2 The effect of siMMR on the interference of MLH1, MSH2, and MSH6 protein expression in CT26 cells was investigated. The results showed that siMMR effectively interfered with the expression of MLH1, MSH2, and MSH6 proteins in CT26 cells, with silencing efficiencies of 43%, 60%, and 75%, respectively.

[0128] Example 4: The interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 mRNA in HCT116 cells. This embodiment investigates the interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 mRNA in HCT116 cells. The specific experimental steps are as follows: siMMR was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and HCT116 cells were purchased from ATCC.

[0129] Step 4.1: HCT116 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and placed in a cell culture incubator with 5% CO2, 37°C and saturated humidity. The cells were then passaged using 0.25% trypsin.

[0130] Step 4.2: Take HCT116 cells from passages 8-10, digest and count them, then divide them into groups of 1×10⁶ cells per well. 5 Cells were seeded at a density of 70%-80% in 12-well plates. After 24 hours, when the cell density reached 70%-80%, siRNA transfection was performed. Three control groups, NC siRNA groups, and siMMR groups were included, with each group having three parallel wells for biological replicates.

[0131] Step 4.3: Perform transfection according to the Lipofectamine™ 3000 (Lipo3000) transfection reagent operation guide. The final concentration of si-NC and each siRNA for transfection is 50 nM.

[0132] Step 4.4: After 48 h of transfection incubation, total RNA was extracted from the cells according to the instructions of the iQTMSYBR® Green Supermix RNA Extraction Kit. The obtained RNA was dissolved in 20 µL of RNase-free water, and the RNA content and purity of each treatment group were measured on a nucleic acid protein detector. Using 1 µg of total RNA as a template, cDNA was synthesized according to the instructions of the iScript™ cDNA Synthesis Kit, and quantitative qPCR detection of the target genes MLH1, MSH2, and MSH6 mRNA was performed according to the instructions of the iQTMSYBR® Green Supermix.

[0133] Experimental results are as follows Figure 3 As shown. Figure 3 The study investigated the interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 mRNA in HCT116 cells. Results showed that siMMR effectively interfered with the expression of MLH1, MSH2, and MSH6 mRNA in HCT116 cells, achieving silencing efficiencies of 56%, 47%, and 74% after transfection, respectively.

[0134] Example 5: The interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 mRNA in DLD1 cells. This embodiment investigates the interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 mRNA in DLD1 cells (human colorectal adenocarcinoma epithelial cell line). The specific experimental steps are as follows: MMR siRNAs were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and DLD1 cells were purchased from ATCC.

[0135] Step 5.1: DLD1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and placed in a cell culture incubator with 5% CO2, 37°C and saturated humidity. The cells were then passaged using 0.25% trypsin.

[0136] Step 5.2: Take DLD1 cells from passages 8-10, digest and count them, then divide them into 1×10⁶ cells per well. 5 Cells were seeded at a density of 70%-80% in 12-well plates. After 24 hours, when the cell density reached 70%-80%, siRNA transfection was performed. A blank control group (Control), NC siRNA, and siMMR were included, with three parallel wells in each group for biological replicates.

[0137] Step 5.3: Perform transfection according to the Lipofectamine™ 3000 (Lipo3000) transfection reagent operation guide. The final concentration of si-NC and each siRNA for transfection is 50 nM.

[0138] Step 5.4: After 48 h of transfection incubation, total RNA was extracted from the cells according to the instructions of the iQ™ BioRNA Extraction Kit. The obtained RNA was dissolved in 20 µL of RNase-free water, and the RNA content and purity of each treatment group were measured on a nucleic acid protein detector. Using 1 µg of total RNA as a template, cDNA was synthesized according to the instructions of the iScript™ cDNA Synthesis Kit, and quantitative qPCR detection of the target genes MLH1, MSH2, and MSH6 mRNA was performed according to the instructions of the iQ™ SYBR® Green Supermix.

[0139] Experimental results are as follows Figure 4 As shown. Figure 4 The study investigated the interference effect of siMMR on the expression of MLH1, MSH2, and MSH6 mRNA in DLD1 cells. Results showed that siMMR could interfere with the expression of MLH1, MSH2, and MSH6 mRNA in HCT116 cells, with expression levels of 23%, 13%, and 36% respectively after transfection.

[0140] Example 6 Preparation of siMMR@LNPs Based on the experimental results of Examples 2-5, the human-mouse co-targeting MLH1, MSH2, and MSH6 siRNAs designed in this invention can effectively silence the expression of target genes in mouse and human CRC cells. Therefore, this example further utilizes a lipid nanoparticle nucleic acid delivery vector to achieve in vivo delivery of siRNA, verifying the in vivo silencing efficiency and anti-MSS CRC efficacy of MLH1, MSH2, and MSH6 siRNAs. The prepared LNPs were named NC siRNA@LNP and siMMR@LNPs.

[0141] The preparation method of NC siRNA@LNP and siMMR@LNPs specifically includes the following steps: Step 6.1: Dilute the siRNA to 130 ng / μL with a 0.05 M sodium acetate solution at pH 5.0, which is called the aqueous phase; disodium 4-2-OH, DSPC (distearylphosphatidylcholine), cholesterol, and DMG-PEG 2000 (dimyristicoglycerol-polyethylene glycol 2000) are dissolved in anhydrous ethanol and mixed in a molar ratio of 48.5:10:40:1.5, which is called the alcohol phase, with a total concentration of 10 mg / mL.

[0142] The synthesis of the cationic lipid C8-4-2-OH was based on Chinese invention patent CN202510341167.2. The general synthesis process is as follows: 2-(tert-butyldimethoxy)ethylamine (1 eq.) was dissolved in anhydrous acetonitrile, and N-(4-bromohexyl)phthalimide (2.2 eq.) and anhydrous potassium carbonate (3 eq.) were added. The mixture was refluxed overnight. The reaction solution was cooled to room temperature, and the insoluble matter was filtered off. Water and dichloromethane were added to separate the layers. The organic layer was separated, washed with saturated brine, and dried over anhydrous sodium sulfate. The drying agent was filtered off, the mixture was evaporated under reduced pressure, and dissolved in anhydrous ethanol. Hydrazine hydrate (10 eq.) was added, and the mixture was refluxed for 5 h. The reaction solution was cooled to room temperature and diluted with methyl tert-butyl ether. The insoluble matter was filtered off, the mixture was evaporated under reduced pressure, and then 80% saturated sodium sulfate aqueous solution and dichloromethane were added to separate the layers. The organic layer was separated, evaporated under reduced pressure, and dissolved in anhydrous tetrahydrofuran. Octyl acrylate (6 eq.) and DBU (1 eq.) were added, and the reaction was carried out at 50 °C for 4 days. The reaction solution was cooled to room temperature, and water and ethyl acetate were added to separate the layers. The organic layer was separated, washed with saturated brine, and dried over anhydrous sodium sulfate. The desiccant was filtered off, the solution was evaporated under reduced pressure, and purified by silica gel column chromatography to give a pale yellow oily substance C8-4-2-OH.

[0143] The characterization data of the above cationic lipid C8-4-2-OH are as follows: 1H NMR (500 MHz, Chloroform-d) δ4.04 (t, J = 6.8 Hz, 8H), 3.55 (t, J = 5.3 Hz, 2H), 2.76 (t, J = 7.3 Hz, 8H), 2.60 (t, J = 5.3 Hz, 2H), 2.48 (t, J = 6.8 Hz, 4H), 2.45 - 2.39 (m, 12H),1.61 (quintet, J = 6.8 Hz, 8H), 1.45 - 1.38 (m, 8H), 1.37 - 1.25 (m, 40H),0.88 (t, J = 6.8 Hz, 12H).

[0144] Step 6.2: Mix the alcohol phase and the aqueous phase at a volume ratio of 1:3 using a microfluidic instrument, collect the mixture, add 10 times the volume of PBS buffer, and centrifuge and ultrafilter using a 10kDa ultrafiltration tube at 4°C. The resulting product is LNP (i.e., NCsiRNA@LNP or siMMR@LNPs).

[0145] Example 7: Efficacy of siMMR@LNPs combined with oxaliplatin in vivo for anti-MSS CRC This example was used to investigate the efficacy of siMMR@LNPs prepared in Example 6 combined with oxaliplatin in vivo against MSS CRC. The specific experimental steps are as follows: A subcutaneous MSS CRC model was constructed using CT26 cells. The specific construction protocol is as follows: CT26 cells of passage 10 or less were digested and then... 5 BALB / c mice were inoculated with cells at a density of 100 cells / mouse on the right back. The tumor was treated when it grew to 100 cm. 3 Mice were randomly divided into three groups: a placebo PBS group, a negative control NC siRNA@LNP+oxaliplatin group, and a siMMR@LNPs+oxaliplatin group, with five mice in each group. Mice were injected subcutaneously with PBS / NC siRNA@LNP / siMMR@LNPs, 60 μg / mouse, every two days. Mice in the NC siRNA@LNP group and the siMMR@LNPs group were injected intraperitoneally with oxaliplatin, 2 mg / kg, for a total of four injections.

[0146] When the tumor volume reaches 1500 cm³ 3 All mice were euthanized, tumors were removed and weighed, and half of the tumors were then subjected to whole-exome sequencing analysis. The remaining tumors were fixed with paraformaldehyde, routinely embedded in paraffin, and stained with IHC to assess the expression levels of MLH1, MSH2, and MSH6 proteins and the level of immune infiltration in the tumors.

[0147] Results in mice Figures 5-9 As shown.

[0148] Figure 5 This represents the weight of a mouse tumor extracted from an in vitro specimen. Figure 5 As shown, the siMMR@LNPs combined with oxaliplatin group had a 30% reduction in ex vivo tumor weight compared to the control group.

[0149] Figure 6 To analyze the expression of MLH1, MSH2, and MSH6 proteins in tumors using immunohistochemistry, compared with the control group, siMMR@LNPs combined with oxaliplatin reduced the expression of MLH1, MSH2, and MSH6 proteins in the tumor microenvironment, with expression rates of 28%, 27%, and 20%, respectively.

[0150] Figure 7 The results showed that after treatment with siMMR@LNPs combined with oxaliplatin, the number of neoantigens with high affinity for MHC I was 1.5 times that of the control group and 1.4 times that of MHC II.

[0151] Figure 8 To assess the immunogenicity of neoantigens with high affinity for MHC I and MHC II, the results showed that 4 of the neoantigens with high affinity for MHC I (4 / 64) could induce IFNγ secretion, and 3 of the neoantigens with high affinity for MHC II (3 / 56) induced strong IFNγ secretion.

[0152] Figure 9 For immunohistochemical analysis of CD8 in the tumor microenvironment + The results showed that siMMR@LNPs combined with oxaliplatin treatment promoted the T-cell immune response in MSS CRC tumors and the level of GZMB secretion.

[0153] Example 8: Efficacy of siMMR@LNPs combined with oxaliplatin in treating MSS CRC liver metastases This example was used to investigate the efficacy of siMMR@LNPs prepared in Example 6 combined with oxaliplatin in treating MSS CRC liver metastases. The specific experimental steps are as follows: A mouse model of MSS CRC liver metastasis was constructed using CT26-Luc cells. The specific construction protocol is as follows: CT26-Luc cells within the 10th passage were collected, and the cell density was adjusted to 2×10⁻⁶ cells by resuspending them in PBS. 6 Cells / mL. BALB / c mice were anesthetized with isoflurane inhalation, and a small incision was made in the left abdomen to expose the spleen. 100 μL of cell suspension (containing 2 × 10⁶ cells / mL) was slowly injected subcapsularly into the spleen using an insulin injector. 5(1 CT26-Luc cells), after injection, apply pressure to stop bleeding for 1 minute, reposition the spleen, and suture the abdominal wall and skin. After injection, the splenic venous blood flow directly flows into the portal vein and perfuses the liver unidirectionally. In addition, the hepatic sinusoidal microvessels physically trap tumor cells, so the tumor cells mainly colonize the liver, forming liver metastases.

[0154] On day 7 after mouse model establishment, the luminescence intensity of mouse livers was detected using a small animal in vivo imaging system. Mice with stable liver luminescence intensity (i.e., successful modeling) were randomly divided into three groups of four mice each: PBS control group: PBS was injected via the tail vein every 3 days; simultaneously, PBS was injected intraperitoneally every 2 days. NC siRNA@LNP+oxaliplatin group: NC siRNA@LNP (prepared in Example 6) was injected via the tail vein at 60 μg / mouse every 3 days; simultaneously, oxaliplatin was injected intraperitoneally at 2 mg / kg every 2 days. siMMR@LNPs+oxaliplatin group: siMMR@LNPs (a mixture targeting MLH1, MSH2, and MSH6, 20 μg each, totaling 60 μg / mouse) was injected via the tail vein every 3 days; simultaneously, oxaliplatin was injected intraperitoneally at 2 mg / kg every 2 days. All administration volumes were 100 μL, administered three times. Small animal in vivo imaging was performed every 3 days after administration. Mice were injected intraperitoneally with luciferase substrate (150 mg / kg), anesthetized with isoflurane 10 minutes later, and placed in a small animal in vivo imaging chamber to collect bioluminescent signals. The luminescence intensity of the liver region was quantitatively analyzed using Living Image software.

[0155] At the experimental endpoint, mouse hearts, livers, spleens, lungs, and kidneys were collected. The weight of the mouse livers was measured, and blood was collected. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a Mindray BS-240 fully automated biochemical analyzer (Shenzhen Mindray). Liver tissue was fixed in paraformaldehyde and then embedded in paraffin. Routine paraffin-embedded sections were sectioned and stained with hematoxylin and eosin (HE) to assess the degree of MSSCRC liver metastasis. Immunohistochemical analysis was performed on the expression of MLH1, MSH2, and MSH6 proteins and CD8 in liver tumor metastases. + T cell infiltration level.

[0156] Mouse experiment results as follows Figures 10-15 As shown.

[0157] Figure 10 The results showed that the luminescence intensity of mouse liver in the siMMR@LNPs combined with oxaliplatin group was significantly lower than that in the NC siRNA@LNP + oxaliplatin group, by approximately 50%. This indicates that MMR siRNA combined with oxaliplatin can effectively inhibit the growth of MSS CRC liver metastases.

[0158] Figure 11Gross images and weights of mouse livers were shown. Results indicated that multiple white metastatic nodules were visible on the liver surface in the PBS control group and the NC siRNA@LNP + oxaliplatin group, with increased liver volume and hardened texture. In contrast, the livers in the siMMR@LNPs + oxaliplatin group resembled normal livers in appearance, with very few metastatic lesions. Liver weight analysis showed that the liver weight in the siMMR@LNPs + oxaliplatin group was comparable to that in the non-model group, approximately 75% of that in the NC siRNA@LNP + oxaliplatin group.

[0159] Figure 12 The results of HE staining of mouse livers showed that large metastatic foci were visible in liver sections of the PBS control group and the NC siRNA@LNP + oxaliplatin group, with large and deeply stained tumor cell nuclei and destruction of normal liver tissue structure; while only small focal metastases were seen in liver sections of the siMMR@LNPs + oxaliplatin group, and the liver lobule structure was basically intact.

[0160] Figure 13 In mice, serum ALT and AST levels were significantly elevated in the PBS group and the NC siRNA@LNP + oxaliplatin group, indicating liver damage caused by liver metastasis. In contrast, ALT and AST levels in the siMMR@LNPs + oxaliplatin group were within the normal range, showing no significant difference compared to the untreated group. This indicates that siMMR@LNPs combined with oxaliplatin can effectively reverse liver damage caused by tumor liver metastasis.

[0161] Figure 14 To analyze the expression levels of MLH1, MSH2, and MSH6 proteins in liver metastases of MSS CRC using immunohistochemistry, the results showed that the protein expression levels of MLH1, MSH2, and MSH6 in the liver metastases of mice in the siMMR@LNPs+oxaliplatin group were significantly reduced, with expression levels of 40%, 25%, and 37% of those in the control group, respectively, demonstrating that the designed siRNA effectively silenced the target genes.

[0162] Figure 15 For immunohistochemical analysis of CD8+ in mouse MSS CRC liver metastases + T cell infiltration levels showed that CD8+ in liver metastases of mice in the siMMR@LNPs+oxaliplatin group was significantly higher than that in other groups. + The level of T cell infiltration was the highest, approximately three times that of the PBS control group, indicating that MMR silencing successfully recruited CD8+ cells. + Cytotoxic T cells enhanced the local anti-tumor immune response.

[0163] The combined experimental results of Examples 7 and 8 demonstrate that siMMR@LNPs combined with oxaliplatin significantly inhibited the in vivo growth rate of MSS CRC, suppressed the expression of MLH1, MSH2, and MSH6 proteins, induced neoantigen production, and enhanced the tumor immune response. In the MSS CRC liver metastasis model, siMMR@LNPs combined with oxaliplatin significantly inhibited the growth of tumor liver metastases and reversed tumor-induced liver damage. This suggests that the siMMR@LNPs combined with oxaliplatin combination drug provided by this invention offers a novel strategy for the treatment of both primary and metastatic lesions of MSS CRC.

[0164] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An siRNA, comprising a sense strand and an antisense strand, characterized in that, The siRNA includes siRNAs that inhibit the expression of MLH1, MSH2 and / or MSH6; Wherein, the nucleotide sequence of the sense strand of the siRNA that inhibits MLH1 expression is a1) or a2). a1) Any one of the sequences in SEQ ID NO:1-10, SEQ ID NO:63-72, or SEQ ID NO:123-126; a2) is a nucleotide sequence that has at least 60% homology with either a1) and has the same function as the sequence shown in a1); The nucleotide sequence of the sense strand of the siRNA that inhibits MLH2 expression is either b1) or b2). b1) Any one of the sequences in SEQ ID NO:11-20, SEQ ID NO:73-82, or SEQ ID NO:127-131; b2) A nucleotide sequence that has at least 60% homology with any one of the sequences in b1) and has the same function as the sequence shown in b1); The nucleotide sequence of the positive strand of the siRNA that inhibits MSH6 expression is c1) or c2). c1) Any one of the sequences in SEQ ID NO:21-30, SEQ ID NO:83-92, or SEQ ID NO:132-138; A nucleotide sequence that has at least 60% homology with either c2) or c1) and has the same function as the sequence shown in c1).

2. The siRNA according to claim 1, characterized in that, The 3' end of the siRNA has a dangling base consisting of two deoxynucleotides; Preferably, the deoxynucleotide is deoxythymidine nucleotide.

3. The siRNA according to claim 1, characterized in that, The siRNA has one or more chemical modifications selected from the following at any site: phosphate thioate (PS) modification, phosphate dithioate (PS2) modification, 2'-O-methyl (2'-OMe) modification, 2'-fluoro (2'-F) modification, 2'-methoxyethyl modification, 2'-O-alkyl modification, 2'-O-allyl modification, 2'-C-allyl modification, 2'-deoxy modification, 2'-hydroxyl modification, 5'-morpholine (5'-Mo) modification, locked nucleic acid (LNA) modification, open-ring nucleic acid (UNA) modification, ethylene glycol nucleic acid (GNA) modification, methylphosphonate (MP) modification, methoxypropylmethylphosphonate (MOP) modification, tricyclic DNA (tcDNA) modification, (S)-restricted ethyl bicyclic nucleic acid ((S)-cEt-BNA) modification, indole modification, peptide nucleic acid (PNA) modification, 5'-(E)-vinyl Phosphonate (VP) modification, N6-methyladenosine (m6A) modification, 5-methylcytidine (m5C) modification, 3-methyluridine (m3U) modification, 5-methylureaside (m5U) modification, pseudoureaside modification, 2-thioureaside (s2U) modification, propynouracil (5-pU) modification, 5'-methylcytosine modification, 5'-ethynyluracil modification, linking the 5' or 3' end of the nucleotide to an inverted abasic nucleotide (invAB) modification, replacing the nucleotide with an inverted abasic nucleotide (invAb) modification, replacing the nucleotide with 2,4-difluorotolyl ribonucleotide (rF) modification or replacing the nucleotide with (S)-glycerol nucleic acid modification, single-chain terminal phosphorylation modification, single-chain terminal cholesterol modification, single-chain terminal galactose modification, single-chain terminal polypeptide modification, single-chain terminal fluorescent probe labeling modification, and ligand modification.

4. A siRNA combination, characterized in that, The siRNA combination includes at least two siRNAs as described in any one of claims 1-3.

5. The siRNA combination according to claim 4, characterized in that, The siRNA combination includes siRNA that inhibits MLH1 expression, siRNA that inhibits MLH2 expression, and siRNA that inhibits MSH6 expression; Preferably, the proportion of each siRNA in the siRNA combination is greater than 0.1%; Preferably, the proportions of the siRNA that inhibits MLH1 expression, the siRNA that inhibits MLH2 expression, and the siRNA that inhibits MSH6 expression in the siRNA combination are all greater than 0.25%.

6. A carrier, characterized in that, The vector is loaded with the siRNA of any one of claims 1-3, or the combination of siRNAs of claim 4 or 5; Preferably, the carrier includes at least one of the following: viral carrier, liposome, lipid nanoparticle, exosome, GalNAc conjugate, cationic polymer, solid-structure lipid carrier, and protein-based nanocarrier.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the siRNA of any one of claims 1-3, the siRNA combination of claim 4 or 5, or the vector of claim 6.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition further includes at least one DNA mutation inducer; Preferably, the DNA mutation inducer includes at least one of a chemical mutagen and a biological mutagen; Preferably, the chemical mutagen includes at least one of alkylating agents, base analogs, intercalating agents, nitrous acid, sodium azide, hydroxylamine, platinum compounds, antimetabolites, topoisomerase inhibitors, and PARP inhibitors; Preferably, the biomutant includes a base editor.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, The pharmaceutical composition also includes combination drugs; Preferably, the combination drugs include antitumor drugs; Preferably, the antitumor drug is selected from at least one of chemotherapy drugs, radiotherapy drugs, photosensitizers, photothermal agents, immunotherapy drugs, or drugs that relieve immunosuppression; Preferably, the immunotherapy drug is selected from at least one of monoclonal antibodies, immune checkpoint inhibitors, immune cells, oncolytic viruses, or tumor vaccines; Preferably, the immunosuppressant is selected from at least one of mercaptopurine, azathioprine, methotrexate, cyclosporine A, tacrolimus, ruxolitinib, rovaxitinib, tripterygium glycoside, or mycophenolate mofetil.

10. The use of the siRNA of any one of claims 1-3, the siRNA combination of any one of claims 4 or 5, the vector of any one of claims 6, or the pharmaceutical composition of any one of claims 7-9 in the preparation of a medicament for the prevention and / or treatment of tumors; Preferably, the tumor comprises a tumor with an intact DNA mismatch repair system; Preferably, the tumor is a cold tumor; Preferably, the tumor includes at least one of solid tumors, soft tissue tumors, hematopoietic tumors, adenomas, or metastatic tumors; Preferably, the drug achieves prevention and / or treatment of tumors through any of the following pathways: (a) Inhibit tumor volume growth; (b) Inhibit tumor weight increase; (c) Inhibit tumor cell growth; (d) Improve tumor treatment response rate; (e) Improve the efficacy of immunosuppressive drugs; (f) Inhibit tumor cell metastasis; (g) Improve the efficacy of chemotherapy drugs; (h) Inhibit tumors by regulating the subject's immune system; (i) Promote or activate the subject's immune system to kill tumors or inhibit tumor growth.

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