Sirna targeting glioblastoma multiforme and use thereof

Abstract

The present application relates to the technical field of biology, and relates to an siRNA targeting glioblastoma multiforme and a use thereof. Provided is an siRNA targeting glioblastoma multiforme. An antisense strand of this siRNA can specifically bind to a target nucleic acid to initiate degradation of the target nucleic acid, wherein the target nucleic acid comprises a pathogenic gene in glioblastoma multiforme, and the pathogenic gene in glioblastoma multiforme comprises a gene encoding ribonucleotide reductase subunit M2 and / or a gene encoding heat shock protein 47. Studies have shown that both the gene encoding the ribonucleotide reductase subunit M2 and the gene encoding the heat shock protein 47 have a significant correlation with the survival of patients with glioblastoma multiforme. The two genes can be used as therapeutic targets for glioblastoma multiforme. The siRNA capable of effectively knocking down the expression of the two genes in the patients with glioblastoma multiforme has great application prospects in the preparation of drugs for preventing and / or treating glioblastoma multiforme.

Description

A siRNA targeting glioblastoma and its application

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411527574.4, filed on October 29, 2024, entitled "An siRNA Targeting Glioblastoma and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to a siRNA targeting glioblastoma and its application, belonging to the field of biotechnology. Background Technology

[0004] Gliomas are among the most common brain tumors, accounting for 30% of central nervous system brain tumors. According to the World Health Organization (WHO) classification system, gliomas are divided into grades I to IV, with grade IV gliomas, also known as glioblastoma multiforme (GBM), being the most malignant. Glioblastomas commonly occur in the brain parenchyma and are diffusely distributed. They are the most common and aggressive type of glioma clinically, with a median survival of only 12–18 months and a 5-year survival rate of less than 5%.

[0005] Currently, the standard treatments for glioblastoma mainly include surgical resection, radiotherapy (RT), and chemotherapy. Among these, temozolomide (TMZ) is the only first-line chemotherapy drug. However, clinical experience has shown that approximately 55% of glioblastoma patients are primary resistant to temozolomide, and another portion of glioblastoma patients inevitably acquire temozolomide resistance during treatment, leading to poor treatment outcomes. Therefore, there is an urgent need for more effective treatments for glioblastoma.

[0006] With advancements in next-generation sequencing technology, our understanding of the molecular basis and overall genome of glioblastoma has deepened, leading many researchers to explore targeted therapies for this disease. However, the presence of the blood-brain barrier and the high heterogeneity of glioblastoma have resulted in limited efficacy for many glioblastoma targeted therapies in clinical trials, failing to achieve significant progress in prolonging overall survival or improving prognosis. Therefore, there is an urgent need to overcome central delivery efficiency and tumor heterogeneity limitations to obtain more effective glioblastoma targeted therapies.

[0007] RNA interference (RNAi) is a recently discovered, ubiquitous gene silencing phenomenon mediated by small interfering RNA (siRNA) and involving specific enzymes. siRNA is a double-stranded RNA molecule composed of 20-25 nucleotides. In the RNAi pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation, thereby inhibiting the expression of specific genes. Theoretically, RNAi can silence all genes; therefore, siRNA can serve as a targeted therapeutic agent, specifically regulating the expression of disease-related genes.

[0008] Effectively knocking down the expression of glioblastoma-related genes in vivo via siRNA would be highly beneficial for targeted therapy of glioblastoma. Furthermore, due to the specificity and effectiveness of siRNA in knocking down disease-related genes, the development of glioblastoma-targeted therapies based on RNAi holds promise for overcoming central nervous system delivery efficiency and tumor heterogeneity. However, naked siRNA is highly susceptible to degradation by nucleases, making the success of RNAi therapy largely dependent on the siRNA vector and delivery method. Traditional siRNA delivery systems, such as lipid nanoparticles, cationic polymers, and viruses, suffer from problems such as easy biodegradation, low biocompatibility, insufficient cyclic stability, and weak targeting ability. Therefore, in addition to developing siRNAs that can effectively knock down the expression of glioblastoma-related genes in vivo, finding a delivery system that can effectively deliver siRNA into the central nervous system, maintain long-term stability and bioactivity in vivo, and possess high biocompatibility is also crucial for targeted therapy of glioblastoma. Summary of the Invention

[0009] To address the aforementioned issues, this application provides a siRNA targeting glioblastoma, wherein the siRNA comprises a sense strand and an antisense strand; the sense strand of the siRNA is at least partially anticomplementary to the antisense strand to form a double-stranded region; the antisense strand of the siRNA is capable of specifically binding to the target nucleic acid through base complementarity pairing to induce the degradation of the target nucleic acid; the target nucleic acid includes a glioblastoma pathogenic gene; the glioblastoma pathogenic gene includes a gene encoding ribonucleotide reductase subunit M2 (RRM2 gene) and / or a gene encoding heat shock protein 47 (HSP47) (Serpinh1 gene).

[0010] In one embodiment of this application, the target nucleic acid includes mRNA encoding ribonucleotide reductase subunit M2 and / or mRNA encoding heat shock protein 47.

[0011] In one embodiment of this application, the pathogenic genes of the glioblastoma also include the gene encoding the epidermal growth factor receptor (EGFR gene), miR-214 and / or the gene encoding the vascular endothelial growth factor receptor (VEGFR gene).

[0012] In one embodiment of this application, the target nucleic acid includes mRNA encoding epidermal growth factor receptor, miR-214, and / or mRNA encoding vascular endothelial growth factor receptor.

[0013] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.9;

[0014] When the pathogenic gene for glioblastoma is a gene encoding heat shock protein 47, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 5, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 10.

[0015] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.9.

[0016] When the pathogenic gene for glioblastoma is the gene encoding heat shock protein 47, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.10.

[0017] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding the epidermal growth factor receptor, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.6;

[0018] When the pathogenic gene for glioblastoma is miR-214, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7;

[0019] When the pathogenic gene of glioblastoma is a gene encoding the vascular endothelial growth factor receptor, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.8.

[0020] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding the epidermal growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.6.

[0021] When the pathogenic gene for glioblastoma is miR-214, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7.

[0022] When the pathogenic gene of glioblastoma is a gene encoding the vascular endothelial growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.8.

[0023] This application also provides a recombinant nucleic acid molecule comprising shRNA; the shRNA comprising the sense strand and the antisense strand of the aforementioned siRNA; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

[0024] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the shRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.14;

[0025] When the pathogenic gene for glioblastoma is a gene encoding heat shock protein 47, the shRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 15.

[0026] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.14;

[0027] When the pathogenic gene for glioblastoma is the gene encoding heat shock protein 47, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.15.

[0028] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding the epidermal growth factor receptor, the shRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 11;

[0029] When the pathogenic gene for glioblastoma is miR-214, the shRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.12;

[0030] When the pathogenic gene for glioblastoma is a gene encoding the vascular endothelial growth factor receptor, the shRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 13.

[0031] In one embodiment of this application, when the pathogenic gene of glioblastoma is a gene encoding the epidermal growth factor receptor, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.11;

[0032] When the pathogenic gene for glioblastoma is miR-214, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.12;

[0033] When the pathogenic gene for glioblastoma is a gene encoding the vascular endothelial growth factor receptor, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.13.

[0034] In one embodiment of this application, the recombinant nucleic acid molecule further comprises a target nucleic acid molecule, a promoter, and / or a functional nucleic acid molecule; the target nucleic acid molecule encodes a nerve center-targeting peptide; and the functional nucleic acid molecule encodes a lysosomal-associated membrane protein.

[0035] In one embodiment of this application, the nerve center targeting peptide includes rabies virus glycoprotein peptide (RVG), glioma targeting peptide (Angiopep-2), apolipoprotein (APOE), and / or cell-penetrating peptide (CPP) capable of penetrating the blood-brain barrier.

[0036] In one embodiment of this application, the promoter includes a cytomegalovirus promoter (CMV promoter), a lentivirus promoter (EF1 promoter), and / or a polymerase III promoter (U6 promoter).

[0037] In one embodiment of this application, the promoter is a cytomegalovirus promoter; the nucleotide sequence of the cytomegalovirus promoter is shown in SEQ ID NO.16.

[0038] In one embodiment of this application, the lysosome-associated membrane proteins include lysosome-associated membrane protein 2B (LAMP2B), transmembrane protein CD63, transmembrane protein CD9, and / or transmembrane protein CD81.

[0039] In one embodiment of this application, the targeting nucleic acid molecule encodes a rabies virus glycoprotein peptide (RVG); the functional nucleic acid molecule encodes lysosome-associated membrane protein 2B; the targeting nucleic acid molecule inserts between the functional nucleic acid molecules to form a chimeric fragment; the nucleotide sequence of the chimeric fragment is shown in SEQ ID NO.17.

[0040] In one embodiment of this application, the recombinant nucleic acid molecule includes a cytomegalovirus promoter, a chimeric fragment, and an shRNA linker fragment connected in series; the shRNA linker fragment includes shRNAs connected in series corresponding to siRNAs targeting mRNAs encoding epidermal growth factor receptor, siRNAs targeting mRNAs encoding vascular endothelial growth factor receptor, siRNAs targeting mRNAs encoding ribonucleotide reductase subunit M2, siRNAs targeting mRNAs encoding heat shock protein 47, and siRNAs targeting miR-214.

[0041] In one embodiment of this application, the nucleotide sequence of the shRNA linker fragment is shown in SEQ ID NO.18.

[0042] This application also provides a recombinant plasmid that expresses the above-mentioned siRNA; or, the recombinant plasmid carries the above-mentioned recombinant nucleic acid molecule.

[0043] In one embodiment of this application, the vector of the recombinant plasmid includes at least one of a viral vector or a non-viral vector; the viral vector includes at least one of a flavivirus vector, a retrovirus vector, a bacteriophage vector, adenovirus vector, adeno-associated virus vector, vaccinia virus vector, hybrid virus vector, baculovirus vector, herpes simplex virus vector, or lentivirus vector; the non-viral vector includes a plasmid vector.

[0044] In one embodiment of this application, the plasmid vector includes pcDNA6.2 plasmid, pLKO.1 plasmid, PGEM-3zf plasmid, PUC19 plasmid and / or PUC57 plasmid.

[0045] In one embodiment of this application, the recombinant plasmid is prepared by: ligating the above-mentioned recombinant nucleic acid molecule with a linearized vector after enzyme digestion to obtain a recombinant plasmid.

[0046] This application also provides an engineered cell whose genome integrates the above-mentioned siRNA; or, whose genome integrates the above-mentioned recombinant nucleic acid molecule; or, whose engineered cell carries the above-mentioned recombinant plasmid.

[0047] In one embodiment of this application, the engineered cells include liver cells.

[0048] In one embodiment of this application, the engineered cells are modified human liver cells.

[0049] This application also provides a small extracellular vesicle, wherein the small extracellular vesicle encapsulates the above-mentioned siRNA; or, the small extracellular vesicle encapsulates the above-mentioned recombinant nucleic acid molecule; or, the small extracellular vesicle encapsulates the above-mentioned recombinant plasmid; or, the small extracellular vesicle is secreted by the above-mentioned engineered cells.

[0050] In one embodiment of this application, the small extracellular vesicles secreted by the engineered cells contain siRNA; the siRNA is obtained by expressing the recombinant nucleic acid molecules in the engineered cells.

[0051] This application also provides the use of the above-mentioned siRNA or the above-mentioned recombinant nucleic acid molecule or the above-mentioned recombinant plasmid or the above-mentioned engineered cell or the above-mentioned small extracellular vesicle in the preparation of a medicament for the prevention and / or treatment of brain tumors of the central nervous system.

[0052] In one embodiment of this application, the drug comprises an inhibitor; the inhibitor comprises the above-mentioned siRNA, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant plasmid, the above-mentioned engineered cells and / or the above-mentioned small extracellular vesicles.

[0053] In one embodiment of this application, the inhibitor is the aforementioned engineered cell.

[0054] In one embodiment of this application, the drug composition further includes pharmaceutically acceptable excipients.

[0055] In one embodiment of this application, the pharmaceutically acceptable excipient further includes a solvent.

[0056] In one embodiment of this application, the central nervous system brain tumor includes a glioma; the glioma includes glioblastoma.

[0057] This application also provides a medicament for the prevention and / or treatment of central nervous system brain tumors, wherein the medicament comprises an inhibitor; the inhibitor comprises the above-mentioned siRNA, the above-mentioned recombinant nucleic acid molecule, the above-mentioned recombinant plasmid, the above-mentioned engineered cells and / or the above-mentioned small extracellular vesicles.

[0058] In one embodiment of this application, the inhibitor is the aforementioned engineered cell.

[0059] In one embodiment of this application, the drug composition further includes pharmaceutically acceptable excipients.

[0060] In one embodiment of this application, the pharmaceutically acceptable excipient further includes a solvent.

[0061] In one embodiment of this application, the central nervous system brain tumor includes a glioma; the glioma includes glioblastoma.

[0062] This application also provides a method for preventing and / or treating brain tumors of the central nervous system, characterized by administering an inhibitor to a subject in need; said inhibitor comprises the aforementioned siRNA or the aforementioned recombinant nucleic acid molecule or the aforementioned recombinant plasmid or the aforementioned engineered cell or the aforementioned small extracellular vesicle. The technical solution of this application has the following advantages:

[0063] 1. This application provides a siRNA targeting glioblastoma, wherein the antisense strand of the siRNA can specifically bind to the target nucleic acid through base complementary pairing to induce the degradation of the target nucleic acid; the target nucleic acid includes the pathogenic gene of glioblastoma; the pathogenic gene of glioblastoma includes the gene encoding ribonucleotide reductase subunit M2 (RRM2 gene) and / or the gene encoding heat shock protein 47 (HSP47) (Serpinh1 gene). Differential gene enrichment analysis and gene pathway enrichment analysis of glioblastoma patients and healthy individuals revealed that the genes encoding ribonucleotide reductase subunit M2 and heat shock protein 47 were significantly associated with the survival of glioblastoma patients. This suggests that the genes encoding ribonucleotide reductase subunit M2 and heat shock protein 47 could serve as therapeutic targets for glioblastoma. Therefore, siRNAs that can effectively knock down the expression of these two genes in glioblastoma patients show great promise in the preparation of drugs for the prevention and / or treatment of central nervous system brain tumors, especially glioblastoma.

[0064] Furthermore, the pathogenic genes of glioblastoma also include the gene encoding the epidermal growth factor receptor (EGFR gene), miR-214, and / or the gene encoding the vascular endothelial growth factor receptor (VEGFR gene). The genes encoding EGFR, miR-214, and VEGFR are all significantly correlated with the survival of glioblastoma patients. Therefore, these genes can serve as therapeutic targets for glioblastoma. Consequently, siRNAs that can effectively knock down the expression of these three genes in glioblastoma patients show great promise in the development of drugs for the prevention and / or treatment of central nervous system brain tumors, especially glioblastoma.

[0065] Further, when the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the positive strand of the siRNA is shown in SEQ ID NO.4, and the nucleotide sequence of the negative strand is shown in SEQ ID NO.9; when the pathogenic gene of glioblastoma is a gene encoding heat shock protein 47, the nucleotide sequence of the positive strand of the siRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the negative strand is shown in SEQ ID NO.10; when the pathogenic gene of glioblastoma is a gene encoding epidermal growth factor receptor, the nucleotide sequence of the positive strand of the siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the negative strand is shown in SEQ ID NO.6; when the pathogenic gene of glioblastoma is miR-214, the nucleotide sequence of the positive strand of the siRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the negative strand is shown in SEQ ID NO.9. As shown in NO.7; when the pathogenic gene of glioblastoma is a gene encoding vascular endothelial growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8. The siRNA can target the pathogenic gene of glioblastoma; studies have shown that the siRNA has high interference efficiency and can significantly knock down the expression of the pathogenic gene of glioblastoma in vivo; furthermore, studies have shown that small extracellular vesicles encapsulating the siRNA can significantly inhibit the growth of glioblastoma in the mouse brain. Therefore, the siRNA has great application potential in the preparation of drugs for the prevention and / or treatment of central nervous system brain tumors, especially glioblastoma.

[0066] 2. This application also provides a recombinant nucleic acid molecule comprising shRNA, a targeting nucleic acid molecule, a promoter, and / or a functional nucleic acid molecule; the shRNA comprises a sense strand of siRNA targeting glioblastoma and an antisense strand of siRNA targeting glioblastoma; the sense and antisense strands are separated by a stem-loop sequence, forming a hairpin structure; the targeting nucleic acid molecule encodes a central nervous system targeting peptide; and the functional nucleic acid molecule encodes a lysosome-associated membrane protein. This application utilizes synthetic biology methods to tandem a central nervous system targeting peptide capable of penetrating the blood-brain barrier, a lysosome-associated membrane protein capable of forming small extracellular vesicles, and shRNA containing siRNA to obtain a gene loop; this gene loop is injected intravenously into the body, utilizing the body's own liver as a bioreaction substrate, enabling in vivo self-assembly to generate small extracellular vesicles encapsulating therapeutic siRNA, achieving precise delivery of glioblastoma RNAi drugs and multi-target combined therapy. Therefore, the recombinant nucleic acid molecule has great application potential in the preparation of drugs for the prevention and / or treatment of central nervous system brain tumors, especially glioblastoma.

[0067] 3. This application provides a small extracellular vesicle (sEV) that encapsulates siRNA targeting glioblastoma. Small extracellular vesicles (sEVs) are small membrane particles (30–150 nanometers in diameter) secreted by cells. As an important means of cell communication, they facilitate intercellular communication by transporting nucleic acids and proteins between neighboring cells. Compared to traditional delivery systems, sEVs, due to their inherent properties, can evade phagocytosis, prolong the in vivo half-life of drugs, and reduce immunogenicity. Delivery of siRNA targeting glioblastoma via small extracellular vesicles offers several advantages: First, it effectively delivers the siRNA to the central nervous system, maintaining its stability and bioactivity in vivo over a long period. Furthermore, it exhibits high biocompatibility, facilitating the inhibition of glioblastoma-causing gene expression and thus exerting an anti-tumor effect. Second, delivery via small extracellular vesicles avoids the problems associated with using exogenous cell-derived extracellular vesicles, such as complex procedures, high costs, and endotoxin content. Third, experiments have demonstrated that injecting the small extracellular vesicles described in this application into mice significantly inhibits the growth of glioblastoma in the mouse brain, demonstrating significant targeted therapy for glioblastoma. Therefore, these small extracellular vesicles show great promise in the preparation of drugs for the prevention and / or treatment of central nervous system brain tumors, particularly glioblastoma. Attached Figure Description

[0068] Figure 1: Association between the gene encoding deoxyribonucleotide reductase (RRM2) and patient survival.

[0069] Figure 2: Association between the gene encoding heat shock protein 47 (HSP47) and patient survival.

[0070] Figure 3: Interference efficiency of siRNA targeting the VEGFR gene.

[0071] Figure 4: Interference efficiency of siRNA targeting the RRM2 gene.

[0072] Figure 5: Interference efficiency of siRNA targeting the Serpinh1 gene.

[0073] Figure 6: Construction map of a multi-target gene loop with five siRNAs tandem.

[0074] Figure 7: Plasmid map of recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214.

[0075] Figure 8: Experimental flowchart for in vitro functional verification of self-assembled sEV-siRNA.

[0076] Figure 9: Effect of self-assembled sEV-siRNA on EGFR gene mRNA (compared to the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0077] Figure 10: Effect of self-assembled sEV-siRNA on VEGFR gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0078] Figure 11: Effect of self-assembled sEV-siRNA on RRM2 gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0079] Figure 12: Effect of self-assembled sEV-siRNA on Serpinh1 gene mRNA (compared with the control group, self-assembled sEV-siRNA significantly reduced the expression of target gene mRNA).

[0080] Figure 13: Effects of self-assembled sEV-siRNA on proteins (epidermal growth factor receptor, vascular endothelial growth factor, deoxyribonucleotide reductase and heat shock protein 47) (compared to the control group, self-assembled sEV-siRNA significantly reduced the surface area of ​​target proteins).

[0081] Figure 14: Small animal in vivo imaging results of mice in different groups.

[0082] Figure 15: Therapeutic effect of gene loop in mice (tail vein injection of gene loop inhibits the growth of intracranial tumors in orthotopic xenograft mice). Detailed Implementation

[0083] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0084] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0085] Experimental Example 1: Identification of therapeutic targets for glioblastoma

[0086] The specific process is as follows:

[0087] Given the rapid proliferation, strong angiogenesis, and immune evasion characteristics of glioblastoma, epidermal growth factor receptor (EGFR), miR-214, and vascular endothelial growth factor (VEGFR) were initially selected as potential therapeutic targets for GBM. Then, the TCGA database was used to screen for therapeutic targets specific to glioblastoma. The specific screening process involved downloading gene information from 10 healthy individuals and 528 glioblastoma patients from the Affymetrix HG-UG133A platform. Differential gene enrichment analysis and gene pathway enrichment analysis were used to identify genes with significant changes in glioblastoma patients. These genes were then correlated with patient survival. The results are shown in Figures 1 and 2. As shown in Figures 1 and 2, the genes encoding ribonucleotide reductase subunit M2 and heat shock protein 47 are significantly associated with the survival of glioblastoma patients. Therefore, the new therapeutic targets for glioblastoma, deoxyribonucleotide reductase (RRM2) and heat shock protein 47 (HSP47, encoded by the Serpinh1 gene), were finally identified.

[0088] Experimental Example 2: Obtaining siRNA targeting glioblastoma

[0089] The specific process is as follows:

[0090] 1. Design

[0091] Using mRNA encoding epidermal growth factor receptor (EGFR), miR-214, vascular endothelial growth factor receptor (VEGFR), ribonucleotide reductase subunit M2 (RRM2), and heat shock protein 47 (HSP47, encoded by the Serpinh1 gene) as target genes, siRNAs targeting EGFR, miR-214, VEGFR, RRM2, and Serpinh1 genes were designed (see Table 1 for details; Table 1 shows the complementary DNA sequences of the sense and antisense strands of the siRNAs). The siRNAs are as follows:

[0092] The siRNA targeting the EGFR gene has the nucleotide sequence of the sense strand as shown in SEQ ID NO.1 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.6. It is named EGFR-siRNA.

[0093] The siRNA targeting miR-214: the nucleotide sequence of the sense strand is shown in SEQ ID NO.2 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.7. It is named miR-214-siRNA.

[0094] The siRNA1 targeting the VEGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.19 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.25, and it is named VEGFR-siRNA1;

[0095] The siRNA2 targeting the VEGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.3 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.8, and it is named VEGFR-siRNA2;

[0096] The siRNA3 targeting the VEGFR gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.20 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.26, and it is named VEGFR-siRNA3;

[0097] The siRNA1 targeting the RRM2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.21 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.27, and it is named RRM2-siRNA1;

[0098] The siRNA2 targeting the RRM2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.4 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.9. It is named RRM2-siRNA2.

[0099] The siRNA3 targeting the RRM2 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.22 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.28, and it is named RRM2-siRNA3;

[0100] The siRNA1 targeting the Serpinh1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.23 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.29, and it is named Serpinh1-siRNA1;

[0101] The siRNA2 targeting the Serpinh1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.24 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.30. It is named Serpinh1-siRNA2.

[0102] The siRNA3 targeting the Serpinh1 gene: the nucleotide sequence of the sense strand is shown in SEQ ID NO.5 and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.10. It is named Serpinh1-siRNA3.

[0103] 2. Screening

[0104] Mix 5 μL of transfection reagent lipo2000 (purchased from Thermo Fisher Scientific) with 100 μL of Opti-MEM (purchased from Thermo Fisher Scientific) and let stand for 5 min to obtain mixture A; dilute different siRNAs to a concentration of 100 pmol with 100 μL of Opti-MEM and let stand for 5 min to obtain mixture B; mix mixture A and mixture B and let stand for 20 min to obtain transfection solutions containing different siRNAs;

[0105] U87 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were used at a rate of 1×10⁻⁶. 5Inoculation was performed at a rate of 1 cell per well into 6-well plates containing 2 mL of DMEM medium (purchased from Thermo Fisher Scientific) with 10% (v / v) fetal bovine serum in each well. The cells were incubated for 24 h in a 5% (v / v) CO2 incubator at 37°C. After 24 h of incubation, blank control, negative control (siNC), EGFR-siRNA experimental group, miR-214-siRNA experimental group, VEGFR-siRNA1 experimental group, VEGFR-siRNA2 experimental group, VEGFR-siRNA3 experimental group, RRM2-siRNA1 experimental group, RRM2-siRNA2 experimental group, RRM2-siRNA3 experimental group, Serpinh1-siRNA1 experimental group, Serpinh1-siRNA2 experimental group, and Serpinh1-siRNA3 experimental group were set up in the 6-well plates, with 4 replicates for each group.

[0106] After setup, add transfection buffer containing different siRNAs to the wells of the experimental groups (EGFR-siRNA group received transfection buffer containing siRNA targeting the EGFR gene, miR-214-siRNA group received transfection buffer containing siRNA targeting the miR-214 gene, VEGFR-siRNA1 group received transfection buffer containing siRNA1 targeting the VEGFR gene, and so on). Add transfection buffer containing siNC to the wells of the negative control group (the nucleotide sequence of the sense strand of siNC is shown in SEQ ID NO. 31, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 31). As shown in NO.32, the blank control group was added with transfection solution without siRNA and cultured in a 5% (v / v) CO2, 37℃ cell culture incubator for 6 h for transfection; after 6 h of transfection, the old cell culture medium was discarded, and 2 mL of fresh DMEM medium containing 10% (v / v) fetal bovine serum was added to each well of a 6-well plate and cultured in a 5% (v / v) CO2, 37℃ cell culture incubator for another 24 h for transfection;

[0107] 24 h after transfection, cell lysis buffer (purchased from Sigma) was added to 6-well plates at a rate of 500 μL per well, and chloroform was added to 6-well plates at a rate of 100 μL per well. The plates were first vortexed vigorously, then placed on ice for 5 min, and finally centrifuged at 12000 rpm and 4°C for 10 min. After centrifugation, the supernatant was collected, and an equal volume of isopropanol was added. The plates were then incubated at room temperature (25°C) for 16 h, and then centrifuged at 12000 rpm and 4°C for 10 min. After centrifugation, the precipitate was collected, and the precipitate was resuspended in 75% (v / v) ethanol to the original volume (i.e., the same volume as the supernatant). The plates were then centrifuged at 12000 rpm and 4°C for 10 min. After centrifugation, the precipitate was collected, yielding mRNA. The extracted mRNA was used as the test sample, and 18S was used as the internal control gene. The expression levels of the target gene in cells after interference with different siRNAs were detected by qPCR (C values ​​were obtained by qPCR). T The measured values ​​normalized the relative levels of mRNA to 18S rRNA, and were then analyzed using 2... -ΔΔCT The expression level of the target gene was determined by the method to verify the interference efficiency of the designed siRNA. The verification results are shown in Figures 3 to 5.

[0108] The specific steps of qPCR are as follows:

[0109] Reverse transcription: Add the reagents to a 200 μL enzyme-free centrifuge tube according to the reaction system in Table 2, vortex to mix, and then place in a PCR instrument to obtain cDNA (see Table 2 for the specific reverse transcription reaction procedure).

[0110] qPCR: Add the reagents to a 200 μL deenzyme-free centrifuge tube according to the reaction system in Table 3, vortex to mix, centrifuge, and then add to a 96-well plate (see Table 3 for the specific qPCR reaction procedure and Table 4 for the primers used in qPCR).

[0111] As shown in Figure 3, among the siRNAs targeting the VEGFR gene, siRNA2 has the best interference efficiency and significantly knocks down the expression of VEGFR mRNA.

[0112] As shown in Figure 4, among the siRNAs targeting the RRM2 gene, siRNA2 has the best interference efficiency and significantly knocks down the expression of RRM2 mRNA.

[0113] As shown in Figure 5, among the siRNAs targeting the Serpinh1 gene, siRNA3 has the best interference efficiency and significantly knocks down the expression of Serpinh1 mRNA.

[0114] Table 1 siRNA and its sequence

[0115] Table 2. Reverse transcription reaction system and reaction procedure

[0116] Table 3 qPCR reaction system and reaction procedure

[0117] Table 4 qPCR primers and their sequences

[0118] Experimental Example 3: Effects of self-assembled sEV-siRNA on target proteins and mRNA in vitro

[0119] The specific process is as follows:

[0120] 1. Construction of recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214

[0121] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.1 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.6, an siRNA targeting the EGFR gene was designed (the nucleotide sequence of the shRNA is shown in SEQ ID NO.11), and named Hsa-siEGFR.

[0122] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.2 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.7, a siRNA targeting miR-214 was designed (the nucleotide sequence of the shRNA is shown in SEQ ID NO.12), and named Anti-miR-214.

[0123] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.3 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.8, an siRNA targeting the VEGFR gene was designed (the nucleotide sequence of the shRNA is shown in SEQ ID NO.13), and named mmu-siVEGFR.

[0124] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.4 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.9, a siRNA targeting the RRM2 gene was designed (the nucleotide sequence of the shRNA is shown in SEQ ID NO.14), and named Hsa-siRRM2.

[0125] Based on the nucleotide sequence of the sense strand as shown in SEQ ID NO.5 and the nucleotide sequence of the antisense strand as shown in SEQ ID NO.10, an siRNA targeting the Serpinh1 gene was designed (the nucleotide sequence of the shRNA is shown in SEQ ID NO.15), and named Hsa-siSerpinh1.

[0126] Chimeric fragments of the cytomegalovirus promoter (CMV Promoter), the gene encoding the rabies virus glycoprotein peptide (RVG), and the gene encoding lysosome-associated membrane protein 2B (LAMP2B), along with Hsa-siEGFR, mmu-siVEGFR, Hsa-siRRM2, Hsa-siSerpinh1, and Anti-miR-214, were sequentially tandem to obtain a multi-target gene loop (the construction map of the multi-target gene loop with five siRNAs tandem is shown in Figure 6; the nucleotide sequence of the CMV Promoter is shown in SEQ ID NO.16; the nucleotide sequence of the chimeric fragment is shown in SEQ ID NO.17; and the nucleotide sequence of the multi-target gene loop is shown in SEQ ID NO.43).

[0127] A multi-target gene loop was synthesized. The multi-target gene loop and the PCDNA6.2 plasmid vector (purchased from Shanghai Jima Co., Ltd.) were double-digested with BamHI and XhoI (purchased from Thermo Fisher Scientific) (digestion system shown in Table 5; digestion at 37℃ for 30 min followed by inactivation at 85℃ for 5 min). After digestion, the products were electrophoresed onto a gel, and the target fragment was recovered from the gel to obtain the double-digested vector and insert fragment. T4 DNA ligase and T4... DNA ligase buffer (purchased from Takara) was used to ligate the double-digested vector and insert fragment (ligation system shown in Table 6, incubation at 16℃ for 14 h for ligation) to obtain the ligation product; the ligation product was transformed into E. coli competent cells DH5α (purchased from Qingke Biotechnology) to obtain the transformation product; the transformation product was streaked on LB agar plates containing 50 μg / mL ampicillin (Amp) (formulation: 5 g / L yeast, 10 g / L peptone, 10 g / L sodium chloride, and 18 g / L agar powder) and incubated at 37℃. After 12 hours, single colonies were picked; each colony was inoculated into 3 mL of LB liquid medium containing 50 μg / mL spectinomycin (formulation: 5 g / L yeast, 10 g / L peptone, and 10 g / L sodium chloride), and cultured at 37°C for 14 hours to obtain bacterial culture; the recombinant plasmid in the bacterial culture was extracted and purified for sequencing. Successful sequencing yielded the recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214 (the plasmid map of the recombinant plasmid is shown in Figure 7, and the nucleotide sequence of the recombinant plasmid is shown in SEQ ID NO.44).

[0128] 2. Effects of self-assembled sEV-siRNA on target proteins and mRNA in vitro

[0129] Following the experimental procedure shown in Figure 8, the control empty vector plasmid and the recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214 were administered via tail vein injection to 6-week-old BALB / c mice (purchased from Jicui Yaokang) at a dose of 10 mg / kg (solvent: 200 μL PBS buffer). The injections were administered twice daily for a total of four injections. Six hours after the last injection, blood plasma was collected from the mice by enucleation, and exosomes (sEV-siRNA, generated by the self-assembly of multi-target gene loops in mice) were extracted from the plasma using ultracentrifugation. U87 cells (purchased from the Shanghai Institute of Cell Biology) were then used at 7.5 × 10⁻⁶ cells per cell line. 6 Cells were inoculated into 6-well plates containing 2 mL of high-glucose medium (purchased from Gibco) with 10% (v / v) fetal bovine serum (FBS) (Novozymes) per well and cultured for 12 h in a 5% (v / v) CO2, 37°C cell culture incubator. After 12 h of culture, 100 μL of high-glucose medium containing 120 μg of exosomes and 2% (v / v) FBS was added to the wells containing U87 cells, and co-incubated in a 5% (v / v) CO2, 37°C cell culture incubator. After co-incubation for 24 h, the expression level of the target gene in the cells was detected by Realtime-PCR, and the results are shown in Figures 9-12. After co-incubation for 48 h, the expression level of the target protein (the protein encoded by the target gene) in the cells was detected by Western blot, and the results are shown in Figure 13. The Realtime-PCR experiment procedure is as follows:

[0130] RNA extraction: Cell lysis buffer (purchased from Takara) was added to 6-well plates at a rate of 1 mL per well. The plates were then incubated on ice for 45 min, with vigorous shaking every 15 minutes to obtain cell lysis products. The cell lysis products were centrifuged at 12000×g at 4℃ for 10 min to remove cell debris. After centrifugation, the supernatant (A) was collected. 200 μL of chloroform was added to supernatant A, followed by vigorous shaking and then incubation on ice for 5 min to separate the layers. Extract the product; centrifuge the extracted product at 12000×g, 4℃ for 15 min. After centrifugation, collect the supernatant B; add 600 μL of isopropanol to supernatant B and mix well, then incubate at -20℃ for 3 h to obtain crude RNA; centrifuge the crude RNA at 12000×g, 4℃ for 20 min. After centrifugation, tilt the centrifuge tube and pour out the supernatant to obtain a centrifuge tube containing white RNA precipitate; prepare 75% (v / v) ethanol with RNase-free water; add 1 mL of... After pouring 75% ethanol into a centrifuge tube containing white RNA precipitate, the tube was inverted to wash the precipitate, then centrifuged at 12000×g and 4℃ for 10 min. After centrifugation, the tube was tilted to pour out the supernatant, and then briefly centrifuged again to obtain a centrifuge tube containing washed RNA precipitate. Excess liquid in the centrifuge tube containing washed RNA precipitate was aspirated with a pipette, the tube was inverted, and the precipitate was dried to obtain a centrifuge tube containing dried RNA precipitate. 30 μL of RNase-free water was added to the centrifuge tube containing dried RNA precipitate to dissolve the precipitate and obtain the dissolved product. The RNA concentration of the dissolved product was measured (the sample needs to be vortexed and briefly centrifuged before measuring the RNA concentration), and the RNA concentration of the dissolved product was adjusted to 1000 ng / μL using RNase-free water to obtain the sample to be tested.

[0131] Reverse transcription: Prepare the reverse transcription reaction system according to Table 7; add the reverse transcription reaction system to the centrifuge tube, vortex to mix, and then place it in the PCR instrument for reverse transcription reaction (reverse transcription reaction program: 42℃, 2min; 37℃, 15min; 85℃, 5s; 4℃, store), to obtain cDNA solution;

[0132] qPCR detection: 18S was used as an internal control, and quantification was performed using double-stranded DNA dye (SYBR Geen); the qPCR reaction system was prepared according to Table 8; the qPCR reaction system was added to the microtube, vortexed and mixed, and then placed in an LC480 real-time PCR instrument for PCR reaction (PCR reaction program: 95℃ for 5 minutes, 95℃ for 30 seconds for 40 cycles, 6℃ for 30 seconds, 72℃ for 1 minute; primers used for qPCR are shown in Table 4).

[0133] As shown in Figures 9-13, compared with the control empty vector plasmid, exosomes obtained by injecting the recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214 into mice significantly reduced the expression levels of EGFR, VEGFR, RRM2, and Serpinh1 proteins and mRNAs in U87 cells. This result indicates that exosomes extracted from mouse plasma have a relatively high interference efficiency on target genes.

[0134] Table 5 Enzyme digestion system

[0135] Table 6 Connection System

[0136] Table 7 Reverse Transcription Reaction System

[0137] Table 8 qPCR reaction system

[0138] Experiment Example 4: Therapeutic Effect of Gene Circuits in Mice

[0139] The specific process is as follows:

[0140] U87-luc cells (purchased from Shanghai Institute of Cell Biology) were used at a rate of 1×10⁻⁶. 6 A single injection volume (5 μL of high-glucose culture medium) was implanted into the intracranial cavity of nude mice (purchased from Jicui Yaokang) (implantation location relative to the following coordinates of the anterior fontanelle: posterior 0.5 mm, lateral 2.5 mm, and intracerebral 3.5 mm) to construct a glioblastoma xenograft model; 7 days after implantation of U87 cells, the size of the tumor was detected using in vivo imaging to ensure successful formation of glioblastoma in the brain. Nude mice carrying glioblastoma were randomly divided into three groups: a blank control group (n=10), a control group (n=10), and a treatment group (n=10). Mice in the treatment group were injected intravenously with the recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214 (solvent: 200 μL PBS buffer) at a dose of 5 mg / kg. Mice in the control group were injected intravenously with the same dose of the control empty vector plasmid (solvent: 200 μL PBS buffer). Mice in the blank control group were injected intravenously with the same volume of PBS buffer. Injections were given every two days for a total of 14 injections. During the period of plasmid injection, in vivo imaging of mice was performed every seven days to detect the size of the tumor. The results are shown in Figures 14 and 15.

[0141] As shown in Figures 14 and 15, compared with the control empty vector plasmid, after intravenous injection of the recombinant plasmid CMV-RVG-siREGFR+VEGFR+RRM2+Serpinh1+anti-214 via tail vein, in vivo imaging of small animals on day 28 showed that the fluorescence intensity of intracranial tumors in the treatment group mice was significantly lower than that in the control group (reduced by approximately 48%). This result indicates that the gene loop can effectively inhibit the progression of intracranial tumors in mice.

[0142] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A siRNA targeting glioblastoma, characterized in that, The antisense strand of the siRNA can specifically bind to the target nucleic acid through complementary base pairing, thereby inducing the degradation of the target nucleic acid; the target nucleic acid includes the pathogenic gene of glioblastoma. The pathogenic genes of the glioblastoma include genes encoding ribonucleotide reductase subunit M2 and / or genes encoding heat shock protein 47. When the pathogenic gene of glioblastoma is a gene encoding ribonucleotide reductase subunit M2, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.9; When the pathogenic gene for glioblastoma is a gene encoding heat shock protein 47, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO. 5, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.

10.

2. The siRNA as described in claim 1, characterized in that, The pathogenic genes of the glioblastoma also include genes encoding epidermal growth factor receptor, miR-214, and / or genes encoding vascular endothelial growth factor receptor.

3. The siRNA as described in claim 2, characterized in that, When the pathogenic gene of glioblastoma is a gene encoding the epidermal growth factor receptor, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.6; When the pathogenic gene for glioblastoma is miR-214, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.7; When the pathogenic gene of glioblastoma is a gene encoding the vascular endothelial growth factor receptor, the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand contains a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.

8.

4. The siRNA as described in claim 3, characterized in that, When the pathogenic gene of glioblastoma is the gene encoding ribonucleotide reductase subunit M2, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.4, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.9; When the pathogenic gene of glioblastoma is the gene encoding heat shock protein 47, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.5, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.

10. When the pathogenic gene of glioblastoma is a gene encoding the epidermal growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.6; When the pathogenic gene for glioblastoma is miR-214, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO.

7. When the pathogenic gene of glioblastoma is a gene encoding the vascular endothelial growth factor receptor, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.

8.

5. A recombinant nucleic acid molecule, characterized in that, The recombinant nucleic acid molecule comprises shRNA; the shRNA comprises the sense strand of the siRNA according to any one of claims 1 to 4 and the antisense strand of the siRNA according to any one of claims 1 to 4; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

6. A recombinant plasmid, characterized in that, The recombinant plasmid expresses the siRNA according to any one of claims 1 to 4; or, the recombinant plasmid carries the recombinant nucleic acid molecule according to claim 5.

7. An engineered cell, characterized in that, The engineered cell's genome is integrated with the siRNA according to any one of claims 1 to 4; or, the engineered cell's genome is integrated with the recombinant nucleic acid molecule according to claim 5; or, the engineered cell carries the recombinant plasmid according to claim 6.

8. A small extracellular vesicle, characterized in that, The small extracellular vesicles encapsulate the siRNA according to any one of claims 1 to 4; or, the small extracellular vesicles encapsulate the recombinant nucleic acid molecule according to claim 5; or, the small extracellular vesicles encapsulate the recombinant plasmid according to claim 6; or, the small extracellular vesicles are secreted by engineered cells according to claim 7.

9. The use of the siRNA according to any one of claims 1 to 4, the recombinant nucleic acid molecule according to claim 5, the recombinant plasmid according to claim 6, the engineered cell according to claim 7, or the small extracellular vesicle according to claim 8 in the preparation of a medicament for the prevention and / or treatment of brain tumors of the central nervous system.

10. A medicament for the prevention and / or treatment of central nervous system brain tumors, characterized in that, The drug comprises an inhibitor; the inhibitor comprises the siRNA of any one of claims 1 to 4, the recombinant nucleic acid molecule of claim 5, the recombinant plasmid of claim 6, the engineered cell of claim 7, or the small extracellular vesicle of claim 8.

11. A method for preventing and / or treating brain tumors of the central nervous system, characterized in that, This includes administering an inhibitor to a subject in need; said inhibitor comprises the siRNA of any one of claims 1 to 4, the recombinant nucleic acid molecule of claim 5, the recombinant plasmid of claim 6, the engineered cell of claim 7, or the small extracellular vesicle of claim 8.

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