Application of improved vaccinia virus Ankara in preparation of medicine for resisting tumors with low ZAP expression

Abstract

The invention provides application of an improved vaccinia virus Ankara (MAV) in preparation of a medicine for resisting tumors with low ZAP expression, and belongs to the technical field of biological medicines. The invention relates to application of MAV in preparation of drugs for preventing and / or treating tumors with low ZAP expression. In view of the fact that MVA targets cancer cells with low expression of ZAP and has good oncolytic property, the invention further provides an anti-cancer drug, and active ingredients of the anti-cancer drug comprise the improved vaccinia virus Ankara and at least one of the following reagents: a reagent for knocking out or inhibiting ZAP gene expression. In-vivo and in-vitro experiment results show that the MAV is used as a selective oncolytic virus to target cancer cells with low expression of ZAP and clear the cancer cells, and a new thought is provided for prevention and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to the application of the modified vaccinia virus Ankara in the preparation of drugs against tumors with low ZAP expression. Background Technology

[0002] Oncolytic viruses are a novel type of anti-tumor drug. As the name suggests, they can target tumor cells, selectively replicate within cells, directly lyse tumor cells, induce anti-tumor immunity, and have minimal killing effect on normal cells. Natural or genetically modified oncolytic viruses can treat tumors through multiple pathways and methods. There are two types of oncolytic viruses: DNA and RNA viruses. More than 20 viruses are being developed for oncolytic virus research, and those currently under development or in clinical trials include herpes simplex virus (HSV), adenovirus (Adv), measles virus (MV), Newcastle disease virus (NDV), parvovirus (PV), vesicular stomatitis virus (VSV), and vaccinia virus. These viruses all exhibit certain tumor-tropism characteristics. Currently approved oncolytic virus products include: T-Vec, a genetically modified HSV-based virus, which was approved by the FDA in 2015 for the treatment of malignant melanoma; Delytact (teserpaturev / G47), a third-generation oncolytic virus developed based on HSV-1, which was approved by the Japanese Ministry of Health, Labour and Welfare (MHLW) for the treatment of malignant glioma; and H101, a recombinant human adenovirus type 5, which was approved in China for the combination therapy of patients with advanced nasopharyngeal carcinoma.

[0003] Poxviruses are a large family of DNA viruses, typically brick-shaped or oval in shape. Unlike other DNA viruses, they replicate in the cytoplasm. There are many members of the poxvirus family, including genera such as orthopoxvirus, goatpoxvirus, vacciniavirus, and fowlpoxvirus. Vacciniaviruses are widely used as oncolytic virus vectors for developing oncolytic viruses due to several advantages: vacciniaviruses replicate in the cytoplasm, preventing their genome from integrating into the host cell's genome, resulting in high safety; the viral particles contain approximately 190kb of genome, providing a large capacity for inserting approximately 25kb of foreign genes without altering their genetic stability; and they can generate an effective immune response. Vacciniaviruses possess the ability to efficiently lyse cancer cells, and genome modification can enhance their safety and efficacy.

[0004] Modified vacciniavirus ankara (MVA) is a member of the vacciniavirus family. It was obtained by passage CVA in chicken embryo fibroblasts (CEF) for more than 570 generations, during which a large number of genes were lost. MVA cannot replicate in most mammalian cells and is mainly used as an expression vector in cancer therapy. Currently under investigation are the following MVA-based oncolytic viruses: MVA-MUC1-IL-2, an MVA expressing human mucin 1 (MUC1) and IL-2, used in combination with first-line chemotherapy drugs for the treatment of advanced non-small cell lung cancer; MVAΔE5R-Flt3L-OX40L, an MVA that deletes the vaccinia E5R gene and expresses two membrane-anchored transgenes, Flt3L and OX40L, which can induce strong anti-tumor immunity when injected into tumors; and MVA-TAA-4-1BBL, an MVA encoding tumor-associated antigens (TAA) and the immunostimulatory ligand 4-1BBL, which can eradicate solid tumors and control distant untreated tumors when treated locally.

[0005] ZAP is a zinc finger antiviral protein that can restrict the replication of RNA viruses, including flaviviruses, HIV, SARS-CoV-2, influenza A virus, and Newcastle disease virus. Subsequent studies have shown that ZAP can also restrict the replication of large DNA viruses HCMV and MVA. The advantages of MVA as an oncolytic agent lie mainly in its excellent ability to express exogenous genes and its ease of genetic manipulation. However, the potential of MVA itself as an oncolytic virus has not been fully explored. ZAP is lacking in most human cancer cells; whether MVA can target ZAP-deficient cancer cells, lyse cancer cells, and eliminate tumors remains unknown. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an application of the modified vaccinia virus Ankara in the preparation of a drug for tumors with low ZAP expression. The modified vaccinia virus Ankara, as a selective oncolytic virus, targets cancer cells with low ZAP expression and can induce apoptosis to a great extent, thereby achieving the oncolytic effect.

[0007] This invention provides the use of the modified vaccinia virus Ankara in the preparation of medicaments for the prevention and / or treatment of tumors with low ZAP expression.

[0008] Preferably, the tumors expressing low levels of ZAP include liver cancer and / or colon cancer.

[0009] Preferably, the cell lines of the liver cancer include Hep3B and / or PLC5.

[0010] Preferably, the colon cancer cell lines include LoVo and / or HCT116.

[0011] This invention provides an anticancer drug, the active ingredient of which includes a modified vaccinia virus Ankara and at least one of the following reagents: a reagent for knocking out or inhibiting the expression of the ZAP gene.

[0012] Preferably, the reagent for knocking out or inhibiting ZAP gene expression includes any one of the following: siRNA, shRNA, sgRNA targeting the ZAP gene, and a gene editing system containing the sgRNA.

[0013] Preferably, the nucleotide sequence of the siRNA is shown in SEQ ID NO:1 to SEQ ID NO:3; and the DNA sequence targeted by the sgRNA is shown in SEQ ID NO:11 to SEQ ID NO:12.

[0014] This invention provides the application of a modified vaccinia virus Ankara combined with a reagent for knocking out or inhibiting ZAP gene expression in the preparation of anticancer drugs.

[0015] Preferably, the reagent for knocking out or inhibiting ZAP gene expression includes any one of the following: siRNA, shRNA, sgRNA targeting the ZAP gene, and a gene editing system containing the sgRNA;

[0016] The nucleotide sequence of the sgRNA is shown in SEQ ID NO:1 to SEQ ID NO:3; the DNA sequence targeted by the sgRNA is shown in SEQ ID NO:11 to SEQ ID NO:12.

[0017] Preferably, the cancer includes at least one of the following: liver cancer, colon cancer, cervical cancer, and lung cancer.

[0018] This invention provides the application of the modified vaccinia virus Ankara in the preparation of drugs for the prevention and / or treatment of tumors with low ZAP expression. Ankara, as a selective oncolytic virus, targets cancer cells with low ZAP expression and can significantly induce apoptosis, thereby achieving oncolytic effects. Experiments show that the expression level of the ZAP gene in Hep3B liver cancer and LoVo colon cancer cells is much lower than that in A549 lung cancer cells and HeLa cervical cancer cells. Infection of cancer cells with low ZAP expression levels with MVA reduces cancer cell activity, indicating that MVA has the characteristic of targeting cells with low ZAP expression, and its oncolytic effect is better than that of cancer cells with high ZAP expression. Furthermore, to clarify the role of ZAP gene expression in the killing of cancer cells by MVA, cancer cells with low ZAP expression were prepared by gene knockout. Compared with control cells, ZAP gene knockout made the cells more sensitive to MVA infection, with higher intracellular viral titers and significantly reduced cell activity. Simultaneously, in vivo mouse solid tumor experiments also showed that MVA administration effectively inhibited tumor growth and had good safety. It is evident that the MVA of this invention has significant advantages in clearing tumors with low ZAP expression, providing a new approach for the development of anti-tumor drugs.

[0019] This invention provides an anticancer drug whose active ingredients include a modified vaccinia virus (AVV) Ankara and at least one of the following reagents: a reagent for knocking out or inhibiting ZAP gene expression. This invention is based on the characteristic of the modified AVV Ankara to target cancer cells with low ZAP expression levels. By reducing ZAP expression in cancer cells at the gene level, it effectively eliminates cancer cells. Experiments show that knocking down ZAP gene expression in cancer cells makes cancer cells that were originally insensitive to MVA infection more sensitive, significantly improving cell clearance rate. Therefore, this invention provides a universal drug treatment method for the prevention and treatment of tumors. Attached Figure Description

[0020] Figure 1 This is a framework diagram for identifying selective oncolytic viruses targeting ZAP gene-deficient human cancers using MVA, as provided by the present invention.

[0021] Figure 2 The results show that MVA induces more significant cell death in cancer cells with low ZAP expression; A and B show the relative expression levels of ZAP mRNA (left) and protein (right) in different cells; C shows the cell viability results after infection with different doses of MVA; D shows the viral titer of MVA in different cells; E shows the relative expression level of caspase-3 at different time points after cell infection.

[0022] Figure 3Knockdown of ZAP improved the oncolytic effect of MVA; A shows the knockdown detection results of siRNA; B shows the cell viability results of cells infected with different doses of MVA after ZAP knockdown; C shows the viral titer results of cells infected with 1 MOI and 10 MOI of MVA after ZAP knockdown; D shows the relative expression level of caspase-3 at different time points after ZAP knockdown.

[0023] Figure 4 The deficiency of ZAP improved the oncolytic effect of MVA, as shown in A. Western blot analysis of A549-ZAP-KO cells; B. Viral titer of MVA in A549 or A549-ZAP-KO cells; and C. Cell viability results after infection with different doses of MVA.

[0024] Figure 5 MVA significantly eliminated tumors with low ZAP expression in vivo. A represents the treatment timeline of the mouse subcutaneous tumor xenograft model; B represents the tumor size detection results in mice after subcutaneous injection of cancer cells; C represents the monitoring results of mouse body weight after injection of Hep3B or LoVo; and D represents the safety experiment results. Detailed Implementation

[0025] This invention provides the use of the modified vaccinia virus Ankara in the preparation of medicaments for the prevention and / or treatment of tumors with low ZAP expression.

[0026] In this invention, low ZAP expression refers to a low expression level of the ZAP gene or ZAP protein. Tumors with low ZAP expression preferably include liver cancer and / or colon cancer. The liver cancer cell lines preferably include Hep3B and / or PLC5. The colon cancer cell lines preferably include LoVo and / or HCT116. In embodiments of this invention, Hep3B and LoVo are low ZAP expression cell lines with a relative expression level not exceeding 1.

[0027] In this invention, the drug is preferably an antiviral drug. The dosage form of the antiviral drug preferably includes an injectable form. The titer of MAV in the injectable form is preferably 1×10⁻⁶. 7 PFU / mL or higher, more preferably (5–100) × 10⁻⁶ 7 PFU / mL. The excipients of the injection preferably include water for injection. This invention does not impose any particular limitation on the preparation method of the injection; any injection preparation method well-known in the art can be used. The preferred dosage of the injection is 200 μL.

[0028] In this invention, the modified vaccinia virus Ankara (MAV) is reported in the prior art (Peng C, et al. Zinc-finger antiviral protein (ZAP) is a restriction factor for replication of modified vaccinia virus Ankara (MVA) in human cells. PLoS Pathog. 2020, 16:e1008845). MAV is an oncolytic virus that kills cancer cells and inhibits the growth of tumors or cancerous tissues by inducing apoptosis. The marker of apoptosis is caspase-3. Since the higher the degree of apoptosis, the higher the caspase-3 gene expression level, the apoptosis-inducing effect of MVA on cancer cells was evaluated by detecting the caspase-3 gene expression level. The results showed that the caspase-3 gene expression level in each cell was largely consistent with the ZAP gene expression level. In addition, this invention also evaluated the anticancer activity of MAV in terms of intracellular MAV titer and cell activity. The results showed that the titer of MVA in Hep3B and LoVo cells was higher than that in A549 and Hela cells. MVA caused minimal damage to primary human embryonic lung cells MRC-5, and the activity of Hep3B and LoVo cells infected with MVA was much lower than that of A549 and Hela cells.

[0029] In one embodiment of the present invention, the expression levels of ZAP in various cancer cells were verified at both the gene and protein levels. The ZAP expression levels in liver cancer and colon cancer were lower than those in cervical cancer and lung cancer. Meanwhile, MAV showed significant advantages in inhibiting cell activity and inducing apoptosis in liver cancer and / or colon cancer. Furthermore, the oncolytic effect was more pronounced with the extension of MAV infection time (24h, 36h, 48h) and the increase of intracellular viral titer (0MOI, 1MOI, 10MOI).

[0030] In another embodiment of the present invention, solid tumor mouse models were constructed by injecting mice with Hep3B and LoVo cell types, respectively. The results showed that MAV administration could effectively inhibit the growth of Hep3B and LoVo cell tumors, and MAV administration had good drug safety. The survival rate and body weight of the mice were not significantly different from those of the control group.

[0031] Given that MAV has the ability to target cancer cells that express low levels of ZAP, this invention provides an anticancer drug whose active ingredients include modified vaccinia virus Ankara and at least one of the following agents: agents that knock out or inhibit ZAP gene expression.

[0032] In this invention, the reagent for knocking out or inhibiting ZAP gene expression preferably includes any one of the following: siRNA, shRNA, sgRNA targeting the ZAP gene, and a gene editing system containing the sgRNA. This invention does not impose any particular limitation on the design method of siRNA, shRNA, or sgRNA; methods for interfering RNA well-known in the art can be used. The nucleotide sequence of the siRNA is preferably shown in SEQ ID NO:1 to SEQ ID NO:3. This invention does not impose any particular limitation on the preparation method of the gene editing system containing the sgRNA; methods for recombinant gene editing systems well-known in the art can be used. The DNA sequence targeted by the sgRNA is shown in SEQ ID NO:11 to SEQ ID NO:12. Experiments have shown that knocking out or inhibiting ZAP gene expression or blocking ZAP protein function can effectively improve the therapeutic effect of MAV on cancer or tumors, broadening its applicability to a wider range of diseases.

[0033] In this invention, the drug is preferably a gene and virus drug. The dosage form of the gene and virus drug preferably includes an injectable form. The titer of MAV in the injectable form is preferably 1×10⁻⁶. 7 PFU / mL or higher, more preferably (5–100) × 10⁻⁶ 7 PFU / mL. The excipients of the injection preferably include water for injection. This invention does not impose any particular limitation on the preparation method of the injection; any injection preparation method well-known in the art can be used. The preferred dosage of the injection is 200 μL / dose.

[0034] This invention provides the application of a modified vaccinia virus Ankara combined with a reagent for knocking out or inhibiting ZAP gene expression in the preparation of anticancer drugs.

[0035] In this invention, the cancer preferably includes at least one of the following: liver cancer, colon cancer, cervical cancer, and lung cancer, more preferably cervical cancer and lung cancer. The types of reagents are the same as those described in the above technical solutions, and will not be repeated here.

[0036] In this invention, a method for preventing and / or treating tumors with low ZAP expression using MAV involves injecting MAV into the body to be treated. The preferred injection titer of the MAV is 1 × 10⁻⁶. 7 PFU / mL or higher, more preferably (5–100) × 10⁻⁶ 7 PFU / mL. The preferred injection dose is 200 μL / injection. The preferred number of MAV injections is 2, more preferably 2, with an interval of 1 day between each injection. The tumor type preferably includes tumors with low ZAP expression, more preferably liver cancer and / or colon cancer.

[0037] In this invention, a method for preventing and / or treating tumors with low ZAP expression is described using MAV and reagents that knock out or inhibit ZAP gene expression. The reagents that knock out or inhibit ZAP gene expression are injected into the organism to be treated, followed by the injection of MAV into the organism. Preferably, the expression level of the ZAP gene or ZAP protein is detected before MAV infection, and infection with MAV occurs only after the expression level of the ZAP gene or ZAP protein has significantly decreased. The MAV infection method is the same as the above-described technical solution and will not be repeated here.

[0038] The following detailed description, in conjunction with embodiments, illustrates the application of the modified vaccinia virus Ankara provided by this invention in the preparation of drugs for tumors expressing low levels of ZAP, but these descriptions should not be construed as limiting the scope of protection of this invention.

[0039] Example 1

[0040] MVA targets cancer cells that express low levels of ZAP and has a good oncolytic effect.

[0041] 1. Cell culture:

[0042] Hep3B, LoVo, PLC5, HCT116, and MRC-5 cell lines were provided by the Cell Resource Center of Peking Union Medical College. HeLa, A549, and DF-1 cell lines were preserved in our laboratory. Cells were cultured in DMEM, DMEM-F12, or MEM-EBSS media supplemented with 10% (v / v) FBS and 1% penicillin / streptomycin.

[0043] 2. Determination of ZAP expression levels in different cancer cells

[0044] The expression level of ZAP in different cancer cells was detected by qRT-PCR. The specific steps are as follows:

[0045] 2.1. RNA Extraction

[0046] Total RNA was extracted using the RNAfast2000 Total RNA Rapid Extraction Kit (Shanghai Feijie). 1 μg of RNA was transcribed into cDNA according to the instructions of the one-step kit.

[0047] 2.2 qRT-PCR detection

[0048] qPCR was performed using the Power SybrVR Green Master Mix kit (Invitrogen Life Technologies, Carlsbad, CA) on an ABI-7500 qPCR instrument (Applied Biosystems, Foster City, CA). The assay conditions were as follows: 0.4 μL upstream primer, 0.4 μL downstream primer, 8.2 μL sterile deionized water, 1 μL cDNA, and 10 μL SYBR Green I fluorescent dye (Merck). The assay program was 95℃ for 30 s; 95℃ for 3 s, 60℃ for 30 s, for 40 cycles.

[0049] Primers include the following types:

[0050] ZAP-F:5'-GATGGAGTGGCCACAGATAT-3'(SEQ ID NO:4);

[0051] ZAP-R:5'-CAGCATCCTGAATCCTAGGT-3' (SEQ ID NO:5);

[0052] GAPDH-F: 5′-AAGGTCGGAGTCAACGGATTTGGT-3′ (SEQ ID NO: 6);

[0053] GAPDH-R: 5'-ACAAAGTGGTCGTTGAGGGCAATG-3' (SEQ ID NO: 7).

[0054] ZAP expression levels vary in different cancer cells. In lung cancer A549 cells and cervical cancer HeLa cells, expression levels are higher than in liver cancer Hep3B cells and colon cancer LoVo cells. Figure 2 (A)

[0055] 2.3 Determination of ZAP protein expression level

[0056] The Western blot experimental procedure is as follows: Discard the original culture medium, wash the cells once with pre-cooled PBS, and lyse them in the wells with 1× cell lysis buffer and 1× PMSF (Beyotime Biotechnology). Sonicate the cell lysates for 1 min to reduce viscosity, boil in a water bath for 10 min, and then dissolve them in 12% NuPAGE Bis-Tris gel (ThermoFisher). Transfer the proteins to a nitrocellulose membrane using the iBlot2 semi-dry transfer system (ThermoFisher). Block the membrane in TBST buffer containing 5% skim milk for 1 h, incubate the primary antibody overnight at 4°C in antibody dilution buffer, wash three times with TBST the next day, and incubate at room temperature with a secondary antibody conjugated to HRP in blocking buffer for 1 h. Detect the ECL signal using enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate (Thermo Fisher Scientific). ZAP antibody was purchased from Proteintech (16820-1-AP), and GAPDH antibody was purchased from Cell Signaling Technology (CST, 2118S). Rabbit and mouse secondary antibodies that bind to HRP were purchased from Cell Signaling Technology.

[0057] ZAP protein expression levels vary in different cancer cells. In lung cancer A549 cells and cervical cancer HeLa cells, expression levels are higher than in liver cancer Hep3B cells and pancreatic cancer LoVo cells. Figure 2 (B)

[0058] 3. Detect the viral titer of MVA in four types of cancer cells.

[0059] MVA was used to infect low-expressing ZAP cancer cells Hep3B and LoVo cells and high-expressing ZAP cancer cells A549 and Hela cells with infection doses of 1 MOI and 10 MOI, respectively. After 24 hours, the supernatant and cells were harvested. The cells and supernatant were collected after three freeze-thaw cycles, and the viral titer on each cell was detected by plaque assay.

[0060] MVA titers were higher in low-expressing ZAP cancer cells Hep3B and LoVo cells than in high-expressing ZAP cancer cells A549 and HeLa cells. Figure 2 (C)

[0061] 4. Cell viability assay

[0062] 5 × 10⁶ cells per well in a 96-well plate 4After treatment, cell viability was assessed using the MTT assay kit (Beyotime). 10 μL of MTT solution was added to each well, and the cells were incubated at 37°C with 5% CO2 for 4 hours. Then, 100 μL of Formazan solution was added to each well, and the cells were incubated at 37°C for 4 hours. The absorbance was measured at 570 nm.

[0063] Cell viability after 48 hours of infection with different doses of MVA was assessed using the MTT assay. MVA caused minimal damage to primary human embryonic lung cells (MRC-5). The viability of Hep3B and LoVo cells infected with MVA was significantly lower than that of A549 and HeLa cells, regardless of whether the dose was low or high. This indicates that MVA targets cells with low ZAP expression and exhibits better oncolytic effects. Figure 2 (D).

[0064] 5. Detection of apoptosis

[0065] As previously reported, widely distributed oncolytic viruses kill cancer cells by inducing apoptosis. Therefore, caspase-3 is a marker gene for apoptosis and is used to detect whether cancer cells infected with MVA have undergone apoptosis. Four types of cancer cells were seeded into 12-well plates. When the cells reached 90% confluence, they were infected with 3 MOI MVA for 2 hours. The medium was discarded, and the cells were washed twice with PBS. The maintenance medium was then replaced. Cells were harvested at 24, 36, and 48 hours, and RNA was extracted. 1 μg of RNA was reverse transcribed into cDNA, and caspase-3 expression was detected according to step 2.2. The primers for qPCR amplification of the caspase-3 gene are as follows:

[0066] Caspase-3-F:5'-GTAGATGGTTTGAGCTGAG-3' (SEQ ID NO:8);

[0067] Caspase-3-R: 5'-CCAGTGGCGTATGGAGAAATG-3' (SEQ ID NO: 9).

[0068] At different time points, caspase-3 expression levels in cancer cells Hep3B and LoVo, which expressed low levels of ZAP, exceeded those in cancer cells HeLa and A549, which expressed high levels of ZAP, indicating that MVA induced more apoptosis in cancer cells expressing low levels of ZAP. Figure 2 (E).

[0069] Example 2

[0070] Knocking down ZAP induced more cell death in cancer cells that highly expressed ZAP using MVA.

[0071] 1. Detecting the knockdown effect of siRNA

[0072] Three pairs of siRNAs specifically targeting ZAP were designed using the IDT RNAi design tool (integrated DNA technology) and synthesized by Sangon Biotech. Cells were transfected with 20 pmol of siRNA in 12-well plates using the JetPrime transfection kit. The transfection reagent mixture was added to the cells, and Western blot analysis was performed as described above after 48 h. The siRNA sequence for targeting ZAP (siZAP) is as follows:

[0073] siRNA1: 5'-cgguucuuucaggcagccaauu-3' (SEQ ID NO: 1);

[0074] siRNA2: 5'-gcagcggucacagaaaauauu-3' (SEQ ID NO: 2);

[0075] siRNA3: 5'-ccgugccuauggaaucuauuu-3' (SEQ ID NO: 3);

[0076] siNC:5'-uucucgaacgugucacguuu-3' (SEQ ID NO: 10).

[0077] All three siRNAs effectively knocked down ZAP protein expression in cancer cells, with siRNA3 showing better targeted knockout in A549 cells and siRNA1 showing better targeted knockout in HeLa cells. Figure 3 (A)

[0078] 2. After knocking down ZAP, the viral titer of MVA in cancer cells was detected.

[0079] HeLa and A549 cells were knocked down with siRNA3 for 48 hours. MVA was then used to infect cancer cells with infection doses of 1 MOI and 10 MOI, respectively. After 24 hours, the supernatant and cells were harvested. The cells and supernatant were collected after three freeze-thaw cycles, and the viral titer on each cell was detected by plaque assay.

[0080] Compared to the siNC group, knocking down ZAP significantly increased viral titers in A549 and HeLa cells, regardless of whether cells were infected with 1 MOI or 10 MOI MVA. Figure 3 (B)

[0081] 3. After knocking down ZAP, the cell viability of cancer cells infected with MVA was detected.

[0082] 5 × 10⁶ cells per well in a 96-well plate 4Once the cells reached 50% confluence, 2.5 pmol of siZAP (siRNA3) or siNC was added to each well. After 48 h, MVA cells were infected with 1 MOI or 10 MOI. Cell viability was assessed using the MTT assay kit (Beyotime). 10 μL of LTT solution was added to each well, and the cells were incubated for 4 h at 37°C with 5% CO2. Then, 100 μL of Formazan lysate was added to each well, and the cells were incubated at 37°C for 4 h. The absorbance was measured at 570 nm.

[0083] Cell viability after 48 hours of infection with different doses of MVA was assessed using the MTT assay. Compared to the siNC group, knockdown of ZAP resulted in lower viability for A549 and HeLa cells, regardless of whether the infection was with low or high doses of MVA. This indicates that knockdown of ZAP improved the oncolytic effect of MVA. Figure 3 (C)

[0084] 4. Detection of apoptosis

[0085] HeLa and A549 cells were seeded into 12-well plates and transfected with 20 pmol of siNC or siZAP (siRNA3) when the cells reached 50% confluence. After 48 h, MVA was used to infect cells for 2 h at a 3 MOI infection dose. The culture medium was discarded, and the cells were washed twice with PBS. The maintenance medium was then replaced. Cells were harvested at 24 h, 36 h, and 48 h, and RNA was extracted. 1 μg of RNA was reverse transcribed into cDNA, and caspase-3 expression was detected by qPCR. The detection conditions were: 0.4 μL upstream primer, 0.4 μL downstream primer, 8.2 μL sterile deionized water, 1 μL cDNA, and 10 μL SYBR Green I fluorescent dye (Merck). The detection program was 95℃ for 30 s; 95℃ for 3 s, 60℃ for 30 s, for 40 cycles. The qPCR amplification primers for the caspase-3 gene are SEQ ID NO:8 and SEQ ID NO:9; the GAPDH primers are SEQ ID NO:6 and SEQ ID NO:7.

[0086] The results showed that at different time points, the expression level of caspase-3 in A549 and HeLa cancer cells transfected with siZAP exceeded that in the control group transfected with siNC. Figure 3 (D). This indicates that knocking down ZAP and MVA can induce more apoptosis in cancer cells.

[0087] Example 3

[0088] To clarify the role of ZAP expression in MVA's ability to kill cancer cells, A549-ZAP-KO cells were generated using CRISPR-Cas9 technology in this embodiment. The specific construction method for A549-ZAP-KO cells using CRISPR-Cas9 technology is as follows:

[0089] Two different sgRNAs were designed to target exon 1 region of human ZAP (5'-ggccgGGATCACCCGATCGG-3', SEQ ID NO:11; 5'-GGATCACCCGATCGGTGGTG-3', SEQ ID NO:12) and inserted into PSP cas 9(BB)-2A-GFP. When A549 cells in the wells reached 60% confluence, they were transfected with the above plasmids and classified by flow cytometry based on GFP expression 48 h post-transfection. GFP-positive cells were serially diluted for clonal selection, and ZAP expression was detected by Sanger sequencing and Western blotting with a ZAP antibody.

[0090] A549 cells transfected with pSpCas9(BB)-2A-GFP containing sgRNA targeting ZAP were used to screen single-cell clones via Western blotting using anti-ZAP and anti-GAPDH antibodies. The knockout effect of ZAP was assessed by Western blotting, and the results are as follows: Figure 4 As shown in Figure A, A549-ZAP-KO cells were successfully generated.

[0091] A549-ZAP-KO cells and A549 cells were infected with MVA at an infection dose of 1 MOI or 10 MOI. The supernatant and cells were harvested after 48 h. The cells were subjected to three freeze-thaw cycles. The viral titer was measured on DF-1 cells using the plaque assay to assess the replication of MVA at different MOIs in A549 or A549-ZAP-KO cells.

[0092] The results are as follows Figure 4 As shown in Figure B, when cells were infected with 1 MOI or 10 MOI, the viral titer in A549-ZAP-KO cells was significantly higher than that in A549 cells.

[0093] To investigate cell viability at different doses of MVA, cells in 96-well plates were infected with 0, 1, or 10 MOI MVA when the plates were 90% confluent. At 2 hours, the original culture medium was discarded, the cells were washed twice with PBS, and replaced with fresh maintenance medium. Cell viability was assessed using the MTT assay after 48 hours.

[0094] See results Figure 4 In C, compared to A549 cells, MVA infection of A549-ZAP-KO cells induced more cell death, and the degree of cell death was dose-dependent.

[0095] Example 4

[0096] MVA can effectively eliminate tumors with low ZAP expression in vivo.

[0097] To evaluate the in vivo antitumor effect of MVA, this embodiment first tested the antitumor effect of MVA, and the experimental protocol is as follows: Figure 5 As shown in Figure A. The therapeutic effect of MVA in different solid tumor models: 100 μL of 3 × 10⁻⁶ mcg / mL. 5 Hep3B or LoVo cells were mixed with Matrigel in a 1:1 ratio and injected into the left posterior dorsal region of 4-week-old female Balc / nu nu. When the tumor size was measured to be 50 mm... 3 At that time, 1×10 was injected into the tumor. 7 PFU in MVA or PBS is administered every other day for a total of two injections. Tumor volume is measured using calipers; the calculation formula is given in Formula I.

[0098] Tumor volume (V) = Tumor length (L) × Tumor width (W) 2 / 2 Formula I.

[0099] The animals were weighed every other day, and euthanized on the 28th day of treatment (in accordance with animal welfare requirements, the tumor size of all experimental animals was less than 2000 mm). 3 (Previously euthanized).

[0100] Consistent with in vitro experiments, such as Figure 5 As shown in Figure B, MVA can effectively reduce the size of Hep3B and LoVo tumors.

[0101] The mice's weight was recorded every other day, and the results were as follows: Figure 5 As shown in Figure C, there was no significant difference in body weight between mice treated with MVA and mice treated with PBS.

[0102] MVA safety testing: The safety of MVA was tested using BALB / c-nu-nu mice. Four-week-old female BALB / c-nu-nu mice received two doses (2 × 10⁻⁶) via tail vein. 7 Mice were injected with PFU via MVA, with the control group receiving PBS injection. Mice were weighed every other day. They were euthanized after 30 days.

[0103] Two doses of 2×10 were administered intravenously to mice via the tail vein. 7 PFU MVA was used to monitor mouse survival rate (left) and body weight (right). Results are as follows: Figure 5 As shown in Figure D, the survival rate of mice injected with MVA or PBS was 100%, and there was no significant difference in body weight over 30 days. The results indicate that MVA effectively inhibited tumor growth compared to the PBS group.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of modified vaccinia virus Ankara in the preparation of drugs for the prevention and / or treatment of tumors with low ZAP expression.

2. The application according to claim 1, characterized in that, The tumors expressing low levels of ZAP include liver cancer and / or colon cancer.

3. The application according to claim 2, characterized in that, The cell lines for liver cancer include Hep3B and / or PLC5.

4. The application according to claim 2, characterized in that, The colon cancer cell lines include LoVo and / or HCT116.

5. An anticancer drug, characterized in that, The active ingredients include modified vaccinia virus Ankara and at least one of the following reagents: reagents that knock out or inhibit ZAP gene expression.

6. The anticancer drug according to claim 5, characterized in that, The reagents for knocking out or inhibiting ZAP gene expression include any one of the following: siRNA, shRNA, sgRNA targeting the ZAP gene, and gene editing systems containing said sgRNA.

7. The anticancer drug according to claim 6, characterized in that, The nucleotide sequence of the siRNA is shown in SEQ ID NO:1 to SEQ ID NO:3; The DNA sequence targeted by the sgRNA is shown in SEQ ID NO:11 to SEQ ID NO:

12.

8. Application of improved vaccinia virus Ankara combined with reagents for knocking out or inhibiting ZAP gene expression in the preparation of anticancer drugs.

9. The application according to claim 8, characterized in that, The reagents for knocking out or inhibiting ZAP gene expression include any one of the following: siRNA, shRNA, sgRNA targeting the ZAP gene, and a gene editing system containing the sgRNA; The nucleotide sequence of the siRNA is shown in SEQ ID NO:1 to SEQ ID NO:3; the DNA sequence targeted by the sgRNA is shown in SEQ ID NO:11 to SEQ ID NO:

12.

10. The application according to claim 9, characterized in that, The cancers include at least one of the following: liver cancer, colon cancer, cervical cancer, and lung cancer.

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