A siRNA capable of inhibiting KRAS gene expression and its application

Abstract

The present invention relates to a siRNA capable of inhibiting the expression of the KRAS gene and its application, and belongs to the field of biotechnology. The present invention provides a siRNA capable of inhibiting the expression of the KRAS gene, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse-complemented to form a double-stranded region; the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1 to 3. Experiments have shown that the siRNAs all have high inhibitory activity on the expression of the KRAS gene. Therefore, the siRNAs have great application prospects in the preparation of drugs for preventing and / or treating diseases associated with KRAS gene mutations (such as colorectal cancer and other cancers).

Description

Technical Field

[0001] The invention relates to siRNA capable of inhibiting KRAS gene expression and application thereof, belonging to the field of biotechnology. Background Art

[0002] KRAS is the most common proto-oncogene in human tumors, and its gene mutation is the most powerful cancer driver discovered so far. Statistics show that cancers driven by KRAS mutations account for about 25% of all human solid tumors. Therefore, the development of inhibitors targeting KRAS has always been a hot topic in the field of anti-cancer drug research and development. However, since the surface of the KRAS protein molecule is very "smooth", it is difficult to find suitable sites for small molecules to bind to, and the KRAS enzyme active site has a very high affinity for guanosine triphosphate (GTP), making it difficult to achieve the classic strategy of inhibiting protein kinases using small molecules that can competitively bind to the enzyme active site on the KRAS protein. Therefore, there is still no significant progress in the development of KRAS inhibitors.

[0003] RNA interference (RNAi) refers to the phenomenon of highly conserved, double-stranded RNA (dsRNA)-induced, efficient and specific degradation of homologous mRNAs. RNAi therapy refers to a treatment method that specifically degrades target mRNAs by small interfering RNAs (siRNAs) that specifically bind to target mRNAs encoding proteins, thereby reducing the expression of disease-related proteins and treating the disease at its root. The emergence of RNAi therapy provides a new idea for solving the "undruggable" target of KRAS protein. Since RNAi therapy can theoretically silence the expression of any gene by degrading mRNA, RNAi therapy can circumvent the limitation that small molecule inhibitors of KRAS protein cannot be developed. However, how to find siRNAs that can effectively inhibit the KRAS gene and how to solve the obstacles to the systemic transmission of siRNA drugs in vivo remain a problem. Summary of the invention

[0004] To solve the above problems, the present invention provides an siRNA capable of inhibiting KRAS gene expression, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NOs. 1 to 3.

[0005] In one embodiment of the present invention, the antisense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NOs. 4 to 6.

[0006] In one embodiment of the present invention, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.4;

[0007] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.5;

[0008] Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.6.

[0009] In one embodiment of the present invention, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.4;

[0010] Alternatively, the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.5;

[0011] Alternatively, the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.3, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.6.

[0012] In one embodiment of the present invention, at least one nucleotide in the sense strand or the antisense strand of the siRNA is a modified nucleotide.

[0013] In one embodiment of the present invention, the modification comprises 2'-sugar ring modification, methoxy modification, fluorination modification, phosphorothioate linkage and / or phosphorodithioate linkage.

[0014] The present invention also provides a recombinant nucleic acid molecule, which comprises shRNA; the shRNA comprises the sense strand of the siRNA and the antisense strand of the siRNA; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

[0015] In one embodiment of the present invention, the shRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in any one of SEQ ID NOs. 21 to 23.

[0016] In one embodiment of the present invention, when 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.4, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.21;

[0017] When the nucleotide sequence of the sense strand of the siRNA is as shown in SEQ ID NO.2, and the nucleotide sequence of the antisense strand is as shown in SEQ ID NO.5, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.22;

[0018] When the nucleotide sequence of the sense strand of the siRNA is shown as SEQ ID NO.3 and the nucleotide sequence of the antisense strand is shown as SEQ ID NO.6, the nucleotide sequence of the shRNA is shown as SEQ ID NO.23.

[0019] In one embodiment of the present invention, the recombinant nucleic acid molecule further comprises a promoter, a gene fragment encoding a targeting peptide, a gene fragment encoding a functional protein capable of forming an extracellular vesicle membrane structure, and / or a miRNA skeleton.

[0020] In one embodiment of the present invention, the promoter includes CMV promoter, EF1a promoter and / or SV40 promoter.

[0021] In one embodiment of the present invention, the targeting peptide comprises colorectal cancer targeting peptide TCP-1 (CTPSPFSHC, Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys).

[0022] In one embodiment of the present invention, the functional protein capable of forming an extracellular vesicle membrane structure includes LAMP2B protein (exosome surface protein).

[0023] In one embodiment of the present invention, the miRNA backbone comprises a pre-miR-155 backbone and / or a pre-miR-30 backbone.

[0024] In one embodiment of the present invention, the recombinant nucleic acid molecule includes a promoter, a gene fragment encoding a targeting peptide, a gene fragment encoding a functional protein capable of forming an extracellular vesicle membrane structure, shRNA and a miRNA skeleton connected in series.

[0025] In one embodiment of the present invention, the recombinant nucleic acid molecule comprises a CMV promoter, a gene fragment encoding a colorectal cancer targeting peptide TCP-1, a gene fragment encoding a LAMP2B protein, a shRNA having a nucleotide sequence as shown in SEQ ID NO.21, and a pre-miR-155 skeleton connected in series;

[0026] Alternatively, the recombinant nucleic acid molecule comprises a CMV promoter, a gene fragment encoding a colorectal cancer targeting peptide TCP-1, a gene fragment encoding a LAMP2B protein, a shRNA with a nucleotide sequence as shown in SEQ ID NO.22, and a pre-miR-155 skeleton connected in series;

[0027] The recombinant nucleic acid molecule includes a CMV promoter, a gene fragment encoding a colorectal cancer targeting peptide TCP-1, a gene fragment encoding a LAMP2B protein, a shRNA with a nucleotide sequence as shown in SEQ ID NO.23, and a pre-miR-155 skeleton connected in series;

[0028] The recombinant nucleic acid molecule includes a CMV promoter, a gene fragment encoding a colorectal cancer targeting peptide TCP-1, a gene fragment encoding a LAMP2B protein, a shRNA with a nucleotide sequence as shown in SEQ ID NO.21, a pre-miR-155 skeleton, and a shRNA with a nucleotide sequence as shown in SEQ ID NO.22 and a pre-miR-155 skeleton, which are connected in series in sequence;

[0029] The recombinant nucleic acid molecule includes a CMV promoter, a gene fragment encoding a colorectal cancer targeting peptide TCP-1, a gene fragment encoding a LAMP2B protein, a shRNA with a nucleotide sequence as shown in SEQ ID NO.21, a pre-miR-155 skeleton, and a shRNA with a nucleotide sequence as shown in SEQ ID NO.23 and a pre-miR-155 skeleton, which are connected in series.

[0030] In one embodiment of the present invention, the nucleotide sequence of the recombinant nucleic acid molecule is shown in any one of SEQ ID NOs. 24 to 28.

[0031] The present invention also provides a recombinant plasmid, wherein the recombinant plasmid expresses the above siRNA; or the recombinant plasmid carries the above recombinant nucleic acid molecule.

[0032] In one embodiment of the present invention, 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 phage vector, an adenovirus vector, an adeno-associated virus vector, a vaccinia virus vector, a hybrid virus vector, a baculovirus vector, a herpes simplex virus vector or a lentivirus vector; the non-viral vector includes a plasmid vector.

[0033] In one embodiment of the present invention, the plasmid vector includes pET plasmid, pcDNA plasmid, pGEX plasmid, pUC plasmid and / or BAC plasmid.

[0034] In one embodiment of the present invention, the preparation method of the recombinant plasmid is: the above-mentioned recombinant nucleic acid molecule and the linearized vector are digested and then connected to obtain the recombinant plasmid.

[0035] The present invention also provides an engineered cell, wherein the genome of the engineered cell is integrated with the above siRNA; or, the genome of the engineered cell is integrated with the above recombinant nucleic acid molecule; or, the engineered cell carries the above recombinant plasmid.

[0036] The present invention 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.

[0037] In one embodiment of the present invention, the small extracellular vesicles secreted by the engineered cells contain siRNA; the siRNA is obtained by expressing the recombinant nucleic acid molecule in the engineered cells.

[0038] In one embodiment of the present invention, the small extracellular vesicles include exosomes.

[0039] The present invention also provides the use of the above siRNA or the above recombinant nucleic acid molecule or the above recombinant plasmid or the above engineered cell or the above small extracellular vesicle in the preparation of a drug for preventing and / or treating a disease associated with KRAS gene mutation.

[0040] In one embodiment of the present invention, the disease associated with KRAS gene mutation includes cancer; the cancer includes colorectal cancer, pancreatic cancer, endometrial cancer and / or lung cancer.

[0041] The present invention also provides a drug for preventing and / or treating diseases associated with KRAS gene mutation, wherein the drug 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.

[0042] In one embodiment of the present invention, the disease associated with KRAS gene mutation includes cancer; the cancer includes colorectal cancer, pancreatic cancer, endometrial cancer and / or lung cancer.

[0043] The technical solution of the present invention has the following advantages:

[0044] 1. The present invention provides an siRNA capable of inhibiting the expression of the KRAS gene, wherein the siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse-complemented to form a double-stranded region; the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NOs. 1 to 3. Experiments have shown that the siRNAs all have high inhibitory activity on the expression of the KRAS gene, and therefore, the siRNAs have great application prospects in the preparation of drugs for preventing and / or treating diseases associated with KRAS gene mutations (e.g., colorectal cancer and other cancers).

[0045] 2. The present invention also provides a recombinant plasmid, which is a gene loop carrying a recombinant nucleic acid molecule; the recombinant nucleic acid molecule includes a promoter, a gene fragment encoding a targeting peptide, a gene fragment encoding a functional protein capable of forming an extracellular vesicle membrane structure, shRNA and a miRNA skeleton connected in series; the shRNA contains a sense strand of siRNA and an antisense strand of siRNA; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure; the sense strand of the siRNA contains a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NO.1 to 3. The present invention uses a synthetic biology method to connect a targeting peptide, a functional protein capable of forming an extracellular vesicle membrane structure and an shRNA containing siRNA in series to obtain a gene loop. Animal experiments have confirmed that the gene loop of the present invention can be injected into the body by intravenous injection, and can self-assemble in the body to produce small extracellular vesicles encapsulating therapeutic siRNA, thereby achieving precise delivery of siRNA drugs and solving the obstacles to systemic transmission of siRNA drugs in vivo. Moreover, animal experiments have confirmed that the gene loop of the present invention can effectively inhibit the expression of KRAS protein and tumor progression by intravenous injection into a KRAS mutation-driven colorectal cancer model. This result shows that the gene loop of the present invention has broken through the bottleneck of KRAS being difficult to make into a drug, making it possible to target and treat a variety of cancers driven by KRAS mutations. In addition, animal experiments have confirmed that after the gene loop of the present invention is injected into the body by intravenous injection, the gene loop can use the mammalian liver as a synthetic generator, so that the designed and constructed gene loop expresses siRNA in liver cells, and is wrapped by small extracellular vesicles produced by the liver, and delivered to the whole body via the circulatory system, thereby achieving siRNA delivery outside the liver, and the siRNA expression, encapsulation and delivery process mediated by the gene loop does not bring obvious toxic and side effects. This result shows that the gene loop of the present invention has higher safety than traditional treatment methods. Therefore, the recombinant nucleic acid molecule has great application prospects in the preparation of drugs for preventing and / or treating diseases associated with KRAS gene mutations (such as colorectal cancer and other cancers). BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Schematic diagram of the construction of gene circuits.

[0047] Figure 2 :TCP-1-siR K Representative skeleton maps of gene circuits.

[0048] Figure 3 :Effect of gene circuit on KRAS protein expression level in LS180 cells. Figure 3 A: Western blotting was used to detect the expression level of KRAS protein after transfection of the gene loop in LS180 cells; B: Statistical analysis of the grayscale values ​​of the Western blotting results in Figure A.

[0049] Figure 4 :Effect of gene circuit on KRAS protein expression level in SW480 cells. Figure 4 A: Western blotting was used to detect the expression level of KRAS protein after transfection of the gene loop in SW480 cells; B: Statistical analysis of the grayscale values ​​of the Western blotting results in Figure A.

[0050] Figure 5 :Effect of gene circuit on KRAS protein expression level in HCT116 cells. Figure 5 A: Western blotting was used to detect the expression level of KRAS protein after transfection of the gene loop in HCT116 cells; B: Statistical analysis of the grayscale values ​​of the Western blotting results in Figure A.

[0051] Figure 6 : Schematic diagram of the expression form of the targeting peptide TCP-1 and the construction of the gene loop without the targeting peptide.

[0052] Figure 7 : Efficiency of siRNA expressed in gene loops in LS180 cells detected by Q-PCR. Figure 7 In the figure, A: the content of si-581 expressed by different gene circuits in the exosomes of LS180 cell culture medium; B: the content of si-G12D expressed by different gene circuits in the exosomes of LS180 cell culture medium.

[0053] Figure 8 : Efficiency of siRNA expression in gene loops in HTC116 cells detected by Q-PCR. Figure 8 In the figure, A: the content of si-581 expressed by different gene circuits in the exosomes of HCT116 cell culture medium; B: the content of si-642 expressed by different gene circuits in the exosomes of HCT116 cell culture medium.

[0054] Fig. 9 : Cryosections of tumors from tumor-bearing mice injected with gene loops without targeting peptides.

[0055] Fig.10 : Cryosections of tumors from tumor-bearing mice injected with a targeting peptide-containing gene loop.

[0056] Fig.11 :RNA Scope experiment to detect scrR and siR K -581、TCP-1-siR K -581 Frozen sections of tumors from three groups of mice. Fig.11 In the figure, the red fluorescent signal is siRNA and the green fluorescent signal is KRAS mRNA.

[0057] Fig.12 :The experimental process design diagram for experiments related to the colorectal cancer model constructed with LS180 cells.

[0058] Fig.13 :Statistical curves of survival time of each group of mice in the experiments related to colorectal cancer model constructed with LS180 cells.

[0059] Fig.14 : Tumor tissues of mice in each group in the experiments related to the colorectal cancer model constructed with LS180 cells. Fig.15 In the figure, the dotted circles represent mice that died during the drug administration period.

[0060] Fig.15 : HE staining results of tumor tissues of each group of mice in the experiments related to the colorectal cancer model constructed with LS180 cells.

[0061] Fig.16 : Western blotting detection results of tumor tissues of each group of mice in the experiments related to colorectal cancer model constructed with LS180 cells.

[0062] Fig.17 : Grayscale statistical analysis results of Western blotting detection results of tumor tissues of each group of mice in related experiments on colorectal cancer model constructed with LS180 cells.

[0063] Fig.18 :The experimental process design diagram of the experiments related to the colorectal cancer model constructed with SW480 cells.

[0064] Fig.19 :Statistical curves of survival time of each group of mice in the experiments related to colorectal cancer model constructed with SW480 cells.

[0065] Fig. 20 : Tumor tissues of mice in each group in the experiments related to the colorectal cancer model constructed with SW480 cells. Fig. 22 In the figure, the dotted circles represent mice that died during the drug administration period.

[0066] Fig.21 : HE staining results of tumor tissues of each group of mice in the experiments related to colorectal cancer model constructed with SW480 cells.

[0067] Fig. 22 : Western blotting detection results of tumor tissues of each group of mice in the experiments related to colorectal cancer model constructed with SW480 cells.

[0068] Fig.23 : Grayscale statistical analysis results of Western blotting detection results of tumor tissues of each group of mice in related experiments on colorectal cancer model constructed with SW480 cells.

[0069] Fig.24 : Experimental process design diagram for experiments related to the colorectal cancer model constructed with HCT116 cells.

[0070] Fig.25 : Representative in vivo tumor fluorescence results of each group of mice in the colorectal cancer model experiments constructed with HCT116 cells.

[0071] Fig.26 : Statistical graphs of total fluorescence intensity of each group of mice and quantitative analysis graphs of average fluorescence intensity of tumor sites in experiments related to colorectal cancer models constructed with HCT116 cells.

[0072] Fig. 27 : HE staining results of tumor tissues of each group of mice in the experiments related to the colorectal cancer model constructed with HCT116 cells.

[0073] Fig.28 : Western blotting detection results of tumor tissues of each group of mice in the experiments related to the colorectal cancer model constructed with HCT116 cells.

[0074] Fig.29 : Grayscale statistical analysis results of Western blotting detection results of tumor tissues of each group of mice in related experiments on colorectal cancer model constructed with HCT116 cells.

[0075] Fig.30 : HE staining results of different tissues of each group of mice in healthy mouse related experiments.

[0076] Fig.31 : Blood biochemical indicators of each group of mice in related experiments on healthy mice. Fig.31In the figure, A: the level of PBC in the blood of each group of mice; B: the level of HGB in the blood of each group of mice; C: the level of PLT in the blood of each group of mice; D: the level of WBC in the blood of each group of mice; E: the level of PMBC in the blood of each group of mice; F: the level of NEU in the blood of each group of mice.

[0077] Figure 1 to Figure 31 In the table, *P<0.05; **P<0.01; ***P<0.005; ****P<0.001. DETAILED DESCRIPTION

[0078] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0079] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0080] Example 1: A siRNA capable of inhibiting KRAS gene expression

[0081] This embodiment provides an siRNA capable of inhibiting the expression of the KRAS gene. The nucleotide sequence of the siRNA uses human KRAS mRNA as the target mRNA and is designed for different mutation sites of the KRAS gene, as shown in Table 1 (in Table 1, the base "T" is used to represent the base "U").

[0082] Table 1. siRNAs and their sequences capable of inhibiting KRAS gene expression

[0083]

[0084]

[0085] Example 2: A gene circuit for expressing siRNA

[0086] This embodiment provides a gene loop for expressing siRNA, and the gene loop and its construction method are as follows:

[0087] Gene loop TCP-1-siR K -581:

[0088] According to the si-581 in Example 1, the nucleotide sequence of the sense chain is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense chain is shown in SEQ ID NO.4, shRNA is designed (the nucleotide sequence of shRNA is shown in SEQ ID NO.21); the CMV promoter, the gene fragment encoding the colorectal cancer targeting peptide TCP-1, the gene fragment encoding the LAMP2B protein, the shRNA with the nucleotide sequence shown in SEQ ID NO.21, and the pre-miR-155 backbone are sequentially connected in series to obtain a gene fragment with a nucleotide sequence shown in SEQ ID NO.24; the gene fragment is connected to the pcDNA6.2 vector by homologous recombination to obtain a recombinant plasmid, which is the gene loop TCP-1-siR K -581 (TCP-1-siR containing targeting peptide K The construction process of gene circuit is shown in Figure 1 , TCP-1-siR K Representative skeleton maps of gene circuits are shown in Figure 2 ).

[0089] Gene loop TCP-1-siR K -642: Gene loop TCP-1-siR K Preparation of the isogenic loop TCP-1-siR K -581, the difference is that the shRNA is designed for si-642, the nucleotide sequence of the shRNA is shown in SEQ ID NO.22, and the nucleotide sequence of the gene fragment is shown in SEQ ID NO.25.

[0090] Gene loop TCP-1-siR K -G12D: gene loop TCP-1-siR K Preparation of -G12D isogenic loop TCP-1-siR K -581, the difference is that the shRNA is designed for si-G12D, the nucleotide sequence of the shRNA is shown in SEQ ID NO.23, and the nucleotide sequence of the gene fragment is shown in SEQ ID NO.26.

[0091] Gene loop TCP-1-siR K -294, gene loop TCP-1-siR K -418, gene loop TCP-1-siR K -436, gene loop TCP-1-siR K -518, gene loop TCP-1-siR K -1158, gene loop TCP-1-siR K-3905 and gene loop TCP-1-scrR (gene loop TCP-1-scrR as control): The preparation process of these 7 gene loops is the same as that of gene loop TCP-1-siR K -581, the difference is that the shRNA targets si-294, si-418, si-436, si-518, si-1158, si-3905 and Scrambled siRNA, respectively, and the nucleotide sequences of shRNA are shown in SEQ ID NO.29 to SEQ ID NO.35, respectively.

[0092] Gene loop TCP-1-siR K -581+G12D: gene loop TCP-1-siR K Preparation process of -581+G12D isogenic loop TCP-1-siR K -581, the difference is that the shRNA corresponding to si-581 and the shRNA corresponding to si-G12D are expressed in series, and the nucleotide sequence of the gene fragment is shown in SEQ ID NO.27.

[0093] Gene loop TCP-1-siR K -581+642: Gene loop TCP-1-siR K Preparation process of -581+642 isogenic loop TCP-1-siR K -581, the difference is that the shRNA corresponding to si-581 and the shRNA corresponding to si-642 are expressed in series, and the nucleotide sequence of the gene fragment is shown in SEQ ID NO.28.

[0094] Gene loop scrR, gene loop siR K -581 and gene loop siR K -581+G12D: gene loop scrR, gene loop siR K -581 and gene loop siR K The preparation process of -581+G12D is the same as that of gene loop TCP-1-scrR and gene loop TCP-1-siR K -581 and gene loop TCP-1-siR K -G12D, the difference is that the gene fragment encoding the colorectal cancer targeting peptide TCP-1 is removed (the gene loop scrR is used as a control) (siR without the targeting peptide K The construction process of gene circuit is shown in Figure 6 ).

[0095] Experimental Example 1: Verification of the inhibitory activity of siRNA and its gene circuit

[0096] In this experiment, the constructed TCP-1-siR K The gene circuit was transfected into three common KRAS mutation cancer cells in colorectal cancer: SW480 (KRAS G12V mutation), HCT116 (KRAS G13D mutation), and LS180 (KRAS G12D mutation) to verify its function. The experimental process is as follows:

[0097] A tube of KRAS mutant cancer cells (purchased from the cell bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences) was taken for recovery and inoculated into a T25 cell culture flask supplemented with 5 mL of MEM medium containing 10% (v / v) fetal bovine serum (purchased from Gibco, 10370021), and cultured in a 5% (v / v) CO2, 37°C cell culture incubator until the cell density reached 80%; after the culture, the cultured cells were first digested with trypsin (purchased from Gibco), then the cultured cells were washed with PBS buffer, and then the cultured cells were resuspended with 6 mL of MEM medium containing 10% (v / v) fetal bovine serum to obtain a cell suspension; the cell suspension was inoculated into a 6-well plate with 1 mL of MEM medium containing 10% (v / v) fetal bovine serum added to each well at an inoculation volume of 1 mL / well, and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 16 h until the cell density in each well reached 80% before transfection began.

[0098] 7.5 μL Lipofectamine 2000 transfection reagent (purchased from Invitrogen, 11668500) and 100 μL Opti-MEM medium (purchased from Gibco, 31985070) were mixed and allowed to stand at room temperature (25°C) for 5 min to obtain solution A. 6 μg of gene loop TCP-1-siR K -581、TCP-1-siR K -642、TCP-1-siR K -G12D、TCP-1-siR K -294、TCP-1-siR K -418、TCP-1-siR K -436、TCP-1-siR K -518、TCP-1-siR K -1158、TCP-1-siR K -3905 and TCP-1-scrR (TCP-1-scrR as control) were mixed with 100 μL opti-MEM culture medium to obtain cells containing different TCP-1-siR K Solution B of the gene loop; Solution A was mixed with different TCP-1-siR KThe B solution of the gene loop was mixed and left to stand at room temperature (25°C) for 20 min to obtain the solution containing different TCP-1-siR K Transfection solution for gene circuit.

[0099] Take the 6-well plate with KRAS mutant cancer cells, discard the liquid in the well, add 2 mL / well of opti-MEM medium to the 6-well plate, and add different TCP-1-siR K The transfection solution of the gene loop was added to different wells of a 6-well plate, mixed, and cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 6 h to transfect the cells in the wells; after the transfection, the liquid in the wells was discarded, and MEM culture medium containing 2% (v / v) fetal bovine serum was added to the 6-well plate at an addition volume of 2 mL / well, and continued to be cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 36 h; after the culture, the liquid in the wells was discarded, and the cells were collected for Western blotting detection (GAPDH protein was used as an internal reference for Western blotting detection, and the antibody used was KRAS Polyclonal antibody, purchased from proteintech, 12063-1-AP), different TCP-1-siR K The relative expression of KRAS protein and internal reference protein GAPDH in KRAS mutant cancer cells after gene circuit intervention was expressed by grayscale value analyzed by Image J software. After data processing by Excel software, Graphpad prism software was used to analyze data significance and draw graphs. The relative expression of KRAS protein was used to evaluate the efficacy of different TCP-1-siR K The inhibitory effect of the gene circuit on KRAS protein.

[0100] The results are as follows Figure 3 to Figure 5 As shown, in LS180 cells, TCP-1-siR K -581、TCP-1-siR K -G12D two gene loops have the best inhibitory effect on KRAS protein; TCP-1-siR K -581、TCP-1-siR K The two gene loops of TCP-1-siR-642 have the best inhibitory effect on KRAS protein. Therefore, si-581 and si-G12D are connected in series as core expression elements to make one gene loop express two siRNAs at the same time. Similarly, si-581 and si-642 are also connected in series in the gene loop to form TCP-1-siR K -581+G12D and TCP-1-siR K -581+642 gene loop.

[0101] Experimental Example 2: Validation of siRNA and its targeting peptides for gene loops

[0102] The targeting peptide TCP-1 is a short peptide of only 9 amino acids. After being incorporated into the gene loop, it is transcribed by the CMV promoter. TCP-1 is fused with the lamp2b protein that forms the extracellular vesicle membrane structure, so that the targeting peptide TCP-1 is located on the surface of the exosomes. The exosomes encapsulating siRNA bind to the blood vessels of colorectal cancer tumor tissues, thereby exerting a targeting effect. To verify this process, this experimental example used TCP-1-siR containing the targeting peptide element. K Gene loops and siRs without targeting peptide elements K The gene loops were transfected into LS180 cells and HCT116 cells respectively. The experimental process is as follows:

[0103] Referring to the method of Experimental Example 1, the gene loops TCP-1-scrR and TCP-1-siR containing the targeting peptide were transfected into the KRAS mutant cancer cell LS180. K -581、TCP-1-siR K -581+G12D and gene loops without targeting peptides scrR, siR K -581、siR K -581+G12D, transfected the gene loops TCP-1-scrR and TCP-1-siR containing targeting peptides in KRAS mutant cancer cells HCT116 K -581、TCP-1-siR K -581+642 and gene loops without targeting peptides scrR, siR K -581、siR K -581+642, and after the transfection, the cells were continued to be cultured; after 48 hours of culture, the exosomes in the cell culture medium were collected, and RNA was extracted for Q-PCR detection; wherein, the Q-PCR detection method is as follows:

[0104] 10 μM si-581, si-642 and si-G12D standards were mixed at 10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8After gradient dilution of μM, standard products of different concentration gradients were obtained; the RNA samples and standard products of different concentration gradients were reverse transcribed using the miRNA 1stStrand cDNA Synthesis Kit (by stem-loop) (purchased from Vazyme, MR101) to obtain cDNA of RNA samples and standard products of different concentration gradients;

[0105] The miRNAUniversal SYBR qPCRMasterMix Kit (Vazyme, MQ101) was used to perform Q-PCR on the RNA samples obtained by reverse transcription and the cDNA of the standards with different concentration gradients to obtain the Ct values ​​of the RNA samples and the standards with different concentration gradients; the standard curve was drawn using Excel software according to the test results (with the logarithmic value of the copy number of each standard as the horizontal axis and the Ct value as the vertical axis);

[0106] According to the position of the Ct value of the RNA sample on the standard curve, the exact copy number of si-581, si-642, and si-G12D in the sample was calculated; the siRNA measurement data of the two groups of gene loops containing targeting peptides and those without targeting peptides were analyzed for significance and plotted using Graphpad prism software to compare the efficiency of siRNA delivery by gene loops containing targeting peptides and those without targeting peptides.

[0107] The results are as follows Figure 7-Figure 8 As shown, there is no significant difference in the siRNA content expressed in cells by gene loops containing targeting peptides and those without targeting peptides. This indicates that the introduction of TCP-1 does not interfere with the expression efficiency of siRNA, and there is no significant difference in the siRNA level expressed by the tandem gene loop compared with the gene loop expressing a single siRNA, which means that the expression of two siRNAs on the same vector will not interfere with each other, and it is possible to exert a superimposed interference effect on the target gene.

[0108] Experimental Example 3: Validation of targeting of siRNA and its gene circuit

[0109] In this experiment, siR K -G12D and TCP-1-siR K -G12D gene loops were injected into mice to verify their targeting. The experimental process is as follows:

[0110] Experiment 1:

[0111] The KRAS mutant cancer cell LS180 was lentivirally transfected using the Lenti-EF1α-Luc-T2A-Puro kit (purchased from Beyotime, C4001) to construct the cancer cell LS180-L that stably expresses firefly luciferase. Six BALb / cnude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected, and the cancer cell LS180-L that stably expresses firefly luciferase and Matrigel high-concentration matrix gel (purchased from Biocoat, 354248) were mixed at a volume ratio of 1:4, and 1×10 6 The injection volume of cells was injected into the cecum wall of mice; 21 days after the injection, the size of the tumor was detected using a small animal in vivo imaging device to ensure tumor formation and obtain BALb / c nude mice with successful tumor implantation.

[0112] Six normal BALb / c nude mice without tumor were randomly divided into two groups. K -G12D group and TCP-1-siR K -G12D group, 3 mice in each group; after grouping, siR K -G12D group and TCP-1-siR K -G12D group mice were injected with gene loop siR at a dose of 5 mg / kg in the tail vein K -G12D and TCP-1-siR K -G12D (the solvent was PBS buffer); 12 hours after injection, mouse plasma exosomes were extracted using the Total Exosome Isolation Kit (from plasma) (purchased from Invitrogen, 4484450), and stained with PKH26 to obtain the stained gene loop siR K -G12D exosomes and gene circuit TCP-1-siR after staining K -G12D obtained exosomes; wherein, the labeling method is as follows:

[0113] The exosomes extracted from a single mouse were mixed with 100 μL of solution C to obtain a mixed solution A. 2.5 μL of PKH26 dye solution (purchased from Sigma-Aldrich, PKH26GL-1KT) and 100 μL of C solution to obtain mixed solution B; after mixing mixed solution A and mixed solution B, incubate on ice for 5 minutes, and then add 500 μL of PBS buffer containing 10% (v / v) BSA (filtered through a 0.22 μm filter) to terminate the reaction to obtain a reaction product; after adding 4 mL of 20% (w / v, g / 100 mL) sucrose solution (filtered through a 0.22 μm filter) to a 70Ti ultracentrifuge tube, the reaction product is added to the ultracentrifuge tube, the upper layer is filled with PBS buffer (filtered through a 0.22 μm filter), and centrifuged at 4°C and 120,000 g for 2 hours; after centrifugation, use the same volume of PBS buffer (filtered through a 0.22 μm filter) as the exosomes to resuspend the precipitate to obtain the stained and labeled exosomes.

[0114] Six BALb / c nude mice with successful tumor implantation were randomly divided into two groups: siR K -G12D group and TCP-1-siR K -G12D group, 3 mice in each group; after grouping, siR K -G12D group and TCP-1-siR K -G12D group mice were injected with 150 μL / mouse of the tail vein after dyeing and labeling of gene loop siR K -G12D exosomes and gene circuit TCP-1-siR after staining K -G12D obtained exosomes; 12h after injection, the mice were euthanized, and frozen sections of tumor tissue were taken. The sections were immunofluorescently stained with vascular-specific Marker CD31 (purchased from cell signaling technology, 42777) to characterize the new blood vessels in the tumor. At the same time, an inverted laser confocal microscope was used to observe the immunofluorescently stained tissue sections to detect the distribution of exosomes containing fluorescent signals around tumor blood vessels and the overall distribution of tumor tissue; wherein, the immunofluorescence staining method is as follows:

[0115] The vascular-specific marker CD31 was used as the primary antibody, which was diluted 100 times with a 5% (w / v, g / 100 mL) BSA solution and then added to the slices and incubated at 4°C for 16 h. After the incubation, the slices were washed three times with PBS buffer to obtain slices incubated with the primary antibody. The secondary antibody was a 5% (w / v, g / 100 mL) BSA solution, which was added to the slices incubated with the primary antibody and incubated at room temperature (25°C) for 1 h. After the incubation, an anti-fluorescence quenching sealing agent containing DAPI (purchased from Beyotime, model P0131) was added to seal the slices to complete the immunofluorescence staining of the slices.

[0116] The results are as follows Figure 9-10 As shown, the fluorescence signals of exosomes containing targeting peptides are more enriched around tumor neovascularization, and their overall distribution in the tumor is also significantly increased compared to exosomes without targeting peptides.

[0117] Experiment 2:

[0118] Based on the results of Experiment 1, the BALb / c nude mice with successful tumor implantation were randomly divided into three groups and injected with gene loop siR at a dose of 5 mg / kg into the tail vein. K -G12D、TCP-1-siR K -G12D, scrR (solvent is PBS buffer), injected once every 2 days, euthanized mice after 3 injections, and tumor tissues were frozen and sectioned using RNAscope TM Plus smRNA-RNAHD Reagents Kit (ACD, 322785) uses probes with CY5 dye to label siRNA and use RNAscope TM Plus smRNA-RNA HD Reagents Kit (ACD, 322785) labels KRAS mRNA with a probe containing CY3 dye and detects the content of siRNA in tumor tissue based on the fluorescent signal.

[0119] The results are as follows Fig.11 As shown, the gene circuit TCP-1-siR containing the targeting peptide K -G12D injection group in the tumor tissue siRNA content was significantly higher than the gene loop siR without targeting peptide K -G12D group and scrR control group, and KRAS mRNA level was significantly decreased

[0120] Combining the results of Experiments 1 and 2, it can be seen that the gene loop containing the targeting peptide can successfully express the targeting peptide on the surface of exosomes, allowing the exosomes to target the blood vessels of colorectal cancer tumor tissues, thereby increasing the efficiency of exosomes in delivering KRAS siRNA to tumor tissues.

[0121] Experimental Example 4: Verification of the performance of the gene circuit in vivo for the treatment of colorectal cancer driven by KRAS G12D mutation

[0122] The KRAS mutation rate in colorectal cancer patients is as high as 40.4%, among which the most common mutation type is the mutation of the 12th codon GGT to GAT (i.e., G12D mutation). Therefore, the colorectal cancer cell line LS180 with KRAS G12D mutation was selected to construct a colorectal cancer xenograft model for in vivo verification. The specific process is as follows:

[0123] Referring to Experimental Example 3, LS180 cells and high-concentration Matrigel (purchased from Biocoat, 354248) were mixed at a volume ratio of 1:4 and 1×10 6 The orthotopic model of colorectal cancer was constructed by injecting 100 cells into the cecum wall of mice. According to the experience of the preliminary experiment, the tumor of mice was stably colonized and began to grow rapidly around 18 days after tumor implantation. Therefore, starting from the 18th day, different (TCP-1-siR K -581、TCP-1-siR K -G12D、TCP-1-siR K -581+G12D) gene loop was used for treatment, and the injection was once every 2 days, and the injection dose was 5 mg / kg each time (the solvent was PBS buffer). MTRX1133 is a currently recognized targeted inhibitor of KRAS G12D mutation. Others were used as positive controls and injected intraperitoneally twice a day at a dose of 3 mg / kg (see the experimental process). Fig.12 During the experiment, the survival time of mice in each group was counted; after 3 weeks of continuous treatment, the mice were killed, and the tumor tissues were taken to evaluate the tumor growth by measuring the size of the tumor; the mouse tumor tissues were taken for HE staining; the mouse tumor tissues were taken for Western blotting detection (GAPDH protein was used as the internal reference for Western blotting detection, and the antibody used was KRAS Polyclonal antibody, purchased from proteintech, 12063-1-AP), and Excel and Graphpad prism software were used to analyze the data significance and draw graphs; the relative expression of KRAS protein in mouse tumor tissue after intervention of different gene circuits was expressed by the gray value obtained by Image J software analysis, and the inhibitory effect of different gene circuits on KRAS protein in tumor tissue was evaluated according to the relative expression of KRAS protein.

[0124] The results are as follows Figure 13-14As shown in the figure, whether expressing a single siRNA or two siRNA gene loops, they effectively inhibited tumor growth, and some mice showed tumor regression. K -581+G12D treatment group was the largest; and, repeated survival experiments in the same period showed that these gene circuits significantly prolonged the survival time of mice, and the positive drug MTRX1133 was also able to relatively significantly inhibit tumor progression, but the protective effect on the overall survival of mice was weak, which may be caused by the potential toxic side effects of small molecule chemical drugs such as immunosuppression; HE staining pathological results showed that the tumor cells in the control group (PBS group and scrRNA group) were densely distributed and had high heterogeneity, while the colorectal tumors in the gene circuit treatment group were smaller overall, and most of the tumors had not yet affected the integrity of the mucosal muscular layer and the mucosal layer, suggesting a lower degree of malignancy. In contrast, MTRX1133 treatment did not significantly reduce the size of the tumor; Western blotting experimental detection results showed that gene circuit therapy significantly inhibited the abnormally high expression of KRAS and its activity in tumor tissues.

[0125] Experimental Example 5: Verification of the performance of the gene circuit in vivo for the treatment of colorectal cancer driven by KRAS G12V mutation

[0126] In colorectal cancer, in addition to KRAS G12D mutations, patients also often have KRAS G12V mutations. Therefore, we further explored the therapeutic effect of the gene circuit on colorectal cancer driven by KRAS G12V mutations. The specific process is as follows:

[0127] Referring to Experimental Example 3, SW480 cells and high-concentration Matrigel (purchased from Biocoat, 354248) were mixed at a volume ratio of 1:4 and 1×10 6 The colorectal cancer xenograft model was constructed by injecting 100 cells into the cecum wall of mice. According to preliminary experimental exploration, SW480 cells formed tumors in about 3 weeks. Therefore, starting from the 21st day, different gene loops (TCP-1-siR K -581、TCP-1-siR K -642、TCP-1-siR K -581+642) was used for treatment, and the injection was performed once every 2 days, with each injection dose of 5 mg / kg (the solvent was PBS buffer). Since there is no recognized effective small molecule inhibitor targeting KRAS G12V, 5-Fu, a commonly used chemotherapy drug for colorectal cancer, was selected as a positive control and injected intraperitoneally once every 2 days at a dose of 25 mg / kg (see the experimental process). Fig.18During the experiment, the survival time of mice in each group was counted; after 3 weeks of continuous treatment, the mice were killed, and the tumor tissues were taken to evaluate the tumor growth by measuring the size of the tumor; the mouse tumor tissues were taken for HE staining; the mouse tumor tissues were taken for Western blotting detection (GAPDH protein was used as the internal reference for Western blotting detection, and the antibody used was KRAS Polyclonal antibody, purchased from proteintech, 12063-1-AP), and Excel and Graphpad prism software were used to analyze the significance of the data and draw graphs; the relative expression of KRAS protein in mouse tumor tissue after intervention of different gene circuits was expressed by the gray value obtained by Image J software analysis, and the inhibitory effect of different gene circuits on KRAS protein in tumor tissue was evaluated according to the relative expression of KRAS protein.

[0128] The results are as follows Figure 19 to Figure 23 As shown, the gene loop siR K -581、siR K -642 and siR K -581+642 can significantly inhibit tumor growth; HE staining pathological sections showed that compared with the high density and high heterogeneity of tumor cells in the control group, the tumor cells after gene loop therapy were no longer dense, and showed obvious pathological necrosis in a smaller total area; in addition, it was also observed that gene loop therapy significantly prolonged the survival of mice, and was better than the positive control 5-Fu; Western blotting experimental detection results showed that gene loop therapy significantly inhibited the abnormally high expression of KRAS and its activity in tumor tissues.

[0129] Experimental Example 6: Verification of the performance of the gene circuit in vivo for the treatment of colorectal cancer driven by KRAS G13D mutation

[0130] In colorectal cancer, in addition to KRAS G12D mutations, patients also often have KRAS G13D mutations. Therefore, we further explored the therapeutic effect of the gene circuit on colorectal cancer driven by KRAS G13D mutations. The specific process is as follows:

[0131] Referring to Experimental Example 3, HCT116 cells and high-concentration Matrigel (purchased from Biocoat, 354248) were mixed at a volume ratio of 1:4 and 1×10 6The colorectal cancer xenograft model was constructed by injecting 100 cells into the mouse cecum wall. In order to track the growth status of the tumor more intuitively, the Lenti-EF1α-Luc-T2A-Puro kit (Beyotime, C4001) was used to construct HCT116 cells that stably expressed firefly luciferase to establish an orthotopic model of colorectal cancer in mice. In vivo imaging confirmed that HCT116 cells successfully colonized and maintained stable growth in about 14 to 21 days. Therefore, starting from the 21st day, different gene loops (TCP-1-siR K -581、TCP-1-siR K -642、TCP-1-siR K -581+642) was used for treatment, and the injection was performed once every 2 days, with each injection dose of 5 mg / kg (the solvent was PBS buffer). Since there is no recognized effective small molecule inhibitor targeting KRAS G13D, 5-Fu, a commonly used chemotherapy drug for colorectal cancer, was selected as a positive control and injected intraperitoneally once every 2 days at a dose of 25 mg / kg (see the experimental process). Fig.24 During the experiment, the survival time of mice in each group was counted; after 3 weeks of continuous treatment, the mice were killed, and the tumor tissues were taken to evaluate the tumor growth by measuring the size of the tumor; the mouse tumor tissues were taken for HE staining; the mouse tumor tissues were taken for Western blotting detection (GAPDH protein was used as the internal reference for Western blotting detection, and the antibody used was KRAS Polyclonal antibody, purchased from proteintech, 12063-1-AP), and Excel and Graphpadprism software were used to analyze the data significance and draw graphs; the relative expression of KRAS protein in mouse tumor tissue after intervention of different gene circuits was expressed by the gray value obtained by Image J software analysis, and the inhibitory effect of different gene circuits on KRAS protein in tumor tissue was evaluated according to the relative expression of KRAS protein.

[0132] The results are as follows Figure 25 to Figure 29As shown, on the 42nd day, some mice in the control group and the positive drug group died due to excessive tumor growth or low body weight. Therefore, after the last in vivo imaging, the mice were sacrificed for anatomical analysis of the tumor progression and pathological examination. The statistical results of in vivo fluorescence tracking of mice showed that both the gene loop expressing a single siRNA and the gene loop expressing two siRNAs in tandem effectively inhibited the progression of in situ tumors, but there was no significant difference between the two gene loops. The results of HE pathology combined with immunohistochemistry showed that compared with the control group (PBS group and scrRNA group), the tumor volume in the gene loop treatment group was significantly smaller, and the expression of KRAS protein in the tissue was significantly reduced. Although 5-Fu treatment can significantly inhibit the proliferation activity of tumor cells and affect the disease progression by causing apoptosis and necrosis of tumor cells, its serious toxic side effects still caused the mice to survive poorly, lose weight, and even die. The results of Western blotting experiments showed that gene loop treatment significantly inhibited the abnormally high expression of KRAS and its activity in tumor tissues.

[0133] Experimental Example 7: In vivo safety evaluation of gene circuits

[0134] To further explore the in vivo safety of the gene circuit, healthy BALb / c nude mice were injected with the gene circuit TCP-1-siR via the tail vein, using PBS buffer as a control. K -G12D、TCP-1-siR K -581 and TCP-1-siR K -G12D+581, injected once every 2 days, the dose was 5 mg / kg (solvent was PBS buffer), after 7 injections, the mice were killed, the hematological and biochemical indices of the mice were tested, and the organs of the mice were taken for HE staining analysis.

[0135] The results are as follows Figure 30-31 As shown, the mice treated with the gene loop had normal liver, kidney and myocardial functions and blood routine parameters, just like the mice in the control group (treated with PBS), and no significant changes were found in the pathological examination of tissue sections. However, the MTRX1133 group showed obvious immunological changes, as shown by a decrease in the number of white blood cells (WBC), peripheral blood mononuclear cells (PBMC) and neutrophils (NEU), but the HE staining pathological examination did not show obvious tissue damage. This result shows that the gene loop systemically interferes with the expression of KRAS without causing obvious toxic and side effects. Compared with the immunosuppression that may be caused by traditional small molecule targeted drugs, siRNA drugs are safer.

[0136] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A siRNA capable of inhibiting KRAS gene expression, characterized in that: The siRNA contains a sense strand and an antisense strand; the sense strand and the antisense strand are at least partially reverse-complementary to form a double-stranded region; the sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NOs. 1 to 3.

2. The siRNA according to claim 1, wherein The antisense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in any one of SEQ ID NOs. 4 to 6.

3. The siRNA according to claim 2, characterized in that The sense strand of the siRNA comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.1, and the antisense strand comprises a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO.4; Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.2, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.5; Alternatively, the sense strand of the siRNA comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.3, and the antisense strand comprises a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO.

6.

4. 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 3 and the antisense strand of the siRNA according to any one of claims 1 to 3; the sense strand and the antisense strand are separated by a stem-loop sequence to form a hairpin structure.

5. The nucleic acid molecule according to claim 4, characterized in that The recombinant nucleic acid molecule further comprises a promoter, a gene fragment encoding a targeting peptide, a gene fragment encoding a functional protein capable of forming an extracellular vesicle membrane structure, and / or a miRNA skeleton.

6. A recombinant plasmid, characterized in that: The recombinant plasmid expresses the siRNA according to any one of claims 1 to 3; or, the recombinant plasmid carries the recombinant nucleic acid molecule according to claim 4 or 5.

7. An engineered cell, characterized in that: The genome of the engineered cell is integrated with the siRNA according to any one of claims 1 to 3; or, the genome of the engineered cell is integrated with the recombinant nucleic acid molecule according to claim 4 or 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 described in any one of claims 1 to 3; or, the small extracellular vesicles encapsulate the recombinant nucleic acid molecule described in claim 4 or 5; or, the small extracellular vesicles encapsulate the recombinant plasmid described in claim 6; or, the small extracellular vesicles are secreted by the engineered cells described in claim 7.

9. Use of the siRNA according to any one of claims 1 to 3, the recombinant nucleic acid molecule according to claim 4 or 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 drug for preventing and / or treating a disease associated with a KRAS gene mutation.

10. A drug for preventing and / or treating a disease associated with KRAS gene mutation, characterized in that: The ingredients of the drug include the siRNA according to any one of claims 1 to 3, the recombinant nucleic acid molecule according to claim 4 or 5, the recombinant plasmid according to claim 6, the engineered cell according to claim 7, or the small extracellular vesicle according to claim 8.

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