Method for constructing oncolytic vaccinia virus vector that modulates il-12 expression by means of inducing pyroptosis, and use thereof

Abstract

The present invention relates to a recombinant vaccinia virus vector and the use thereof in the preparation of a drug for treating a tumor. The TK gene and B13 gene are deleted, the GSDME gene, IL-12 gene and GM-CSF gene are inserted, or the IL-18BP gene is deleted in a vaccinia virus to respectively obtain tumor-targeting viral vectors VVΔTK-GSDMEΔB13, VVΔTK-GSDMEΔB13-IL12, VVΔTK-GSDMEΔB13-IL12ΔIL-18BP and VVΔTK-GSDMEΔB13-GMCSF. The VVΔTK-GSDMEΔB13 vector of the present invention provides a method for effectively delivering GSDME to tumor cells and causing pyroptosis of tumor cells, while exhibiting a better tumor inhibitory effect, wherein VVΔTK-GSDMEΔB13-IL12 induces pyroptosis of tumor cells while expressing IL-12, which realizes sustained, stable and relatively low-level expression of IL-12, thereby exerting the anti-tumor effect of IL-12 while reducing the toxicity; VVΔTK-GSD MEΔB13-IL12ΔIL-18BP avoids the inhibitory effect of viral IL-18BP on pyroptosis-produced IL-18, thereby improving the safety and efficacy of synergistic IL-12 and IL-18 treatment; and VVΔTK-GSDMEΔB13-GMCSF improves the anti-tumor efficacy by means of pyroptosis combined with GM-CSF, which provides data support and a new perspective for the combined use of pyroptosis with more therapeutic genes, and shows good application prospects.

Description

Construction Method and Application of Oncolytic Vaccine Virus Vector Regulating IL-12 Expression Through Pyroptosis Induction Technical Field

[0001] This invention relates to a recombinant vaccinia virus vector that delivers GSDME to tumors and induces pyroptosis in tumor cells, and its application in the preparation of tumor drugs, belonging to the fields of genetic engineering and immunotherapy. Background Technology

[0002] Oncolytic virus therapy has proven to be a cancer treatment method with great potential. Oncolytic viruses do not replicate or replicate in very small numbers within normal cells, but they can replicate within tumor cells, directly lysing tumor cells, activating anti-tumor immune responses, regulating angiogenesis and the tumor microenvironment, and recruiting immune cells to infiltrate the tumor, turning it into a "hot tumor." Currently, the main challenges facing the clinical use of oncolytic viruses include poor efficacy as monotherapy and the predominantly intratumoral administration route, which limits their clinical application. Therefore, developing oncolytic viruses with stronger antitumor effects and higher safety profiles that can be administered intravenously and intratumorally is the future direction of viral therapy.

[0003] Intravenous administration is an ideal systemic drug delivery method, meeting the needs of most clinical cancer treatments. Clinical research results show that vaccinia virus (VV) is very suitable for intravenous injection. Furthermore, VV exhibits other advantages, such as its entire infection cycle existing only in the cytoplasm, with viral DNA not integrating into the host genome, resulting in high clinical safety. Additionally, VV has two forms of infection: intracellular membrane virus (IMV) and extracellular enveloped virus (EEV), with the membrane-bound EEV able to evade the body's immune system. Therefore, VV is a promising oncolytic vaccinia virus vector.

[0004] Pyroptosis is an inflammatory programmed cell death process primarily caused by activated caspases or granzymes cleaving Gasdermin (excluding GSDM, GSDMF) family members. This releases the N-terminal domain, creating pores in the cell membrane, causing cell swelling and rupture, leading to cell death and the release of large amounts of inflammatory factors (IL-1β and IL-18) and cellular contents. It also recruits immune cells to amplify the inflammatory response. GSDME, also known as DFNA5, is suppressed in many tumors. When highly expressed in tumor cells, it can be cleaved by activated caspase-3 or granzyme B, releasing its N-terminal domain to create pores in the cell membrane, inducing immunogenic cell death, i.e., pyroptosis. This, in turn, enhances the phagocytic activity of tumor-associated macrophages, increases the infiltration of NK and CD8+ T cells within the tumor, and enhances their cytotoxic function, thus exerting an anti-tumor immune effect. The B13 protein in *Vulpectomyces vulgaris* can inhibit caspase-3 activity. Therefore, deletion of the B13 gene is expected to increase the occurrence of pyroptosis.

[0005] Interleukin-12 (IL-12) has shown great therapeutic potential in tumor immunotherapy. IL-12 is a secreted pro-inflammatory molecule composed of p35 and p40 subunits. It can activate innate and adaptive immunity, enhancing the tumor-killing activity of T cells through pathways such as increased cytotoxicity, proliferation, and Th1 differentiation; it also promotes anti-tumor angiogenesis. However, IL-12 is a double-edged sword; recombinant IL-12 protein can cause lethal toxicity, thus limiting its clinical application. Therefore, reducing the toxicity of IL-12 while maintaining its anti-tumor function is a key research direction for its application.

[0006] Pyroptosis triggers the release of IL-1β and IL-18, but high expression of IL-1β promotes tumor invasion and tumor stemness. IL-18, an inflammatory cytokine and a member of the IL-1 family, primarily functions to induce IFN-γ secretion from T and NK cells. IL-12 and IL-18 have synergistic anti-tumor effects; co-expression of both can reduce IL-12 toxicity without diminishing its anti-tumor activity. The B15 protein expressed by VV is a soluble receptor for IL-1β, while the expressed IL-18-binding protein (IL-18BP, formerly named C12L) inhibits IL-18 function; IL-18BP is a secretory immune checkpoint, weakening the anti-tumor effect of IL-18. Therefore, modifying VV by retaining the B15R gene, deleting the IL-18BP gene, and simultaneously expressing IL-12 may further enhance the anti-tumor activity of a VV vector that deletes the TK and B13 genes and simultaneously expresses GSDME. Therefore, studying the synergistic antitumor effects and safety of IL-12 and pyroptosis will provide new insights for the application of IL-12.

[0007] Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein primarily produced by T cells. GM-CSF can promote anti-tumor immune responses by activating macrophages and dendritic cells, enhancing the immune response of anti-tumor T cells, and inhibiting angiogenesis and endothelial cell migration, thereby reducing tumor growth, angiogenesis, and metastasis. Therefore, inserting oncolytic viruses into the GM-CSF gene can enhance its anti-tumor capabilities. Summary of the Invention

[0008] To address the shortcomings of existing technologies, one objective of this invention is to provide a safe and effective recombinant vaccinia virus vector capable of delivering GSDME to tumors and inducing pyroptosis in tumor cells, and its application in the preparation of therapeutic drugs for tumors.

[0009] The second objective is to provide a safe, effective, and systemically applicable recombinant vaccinia virus vector that regulates IL-12 by inducing pyroptosis and its application in the preparation of antitumor drugs.

[0010] The third objective is to develop a recombinant vaccinia virus vector that enhances the synergistic effect of IL-18 and IL-12 released by pyroptosis through deletion of the IL-18BP gene, and its application in the preparation of antitumor drugs.

[0011] The fourth objective is to provide a recombinant vaccinia virus vector that combines tumor cell pyroptosis with existing therapeutic genes and its application in the preparation of drugs for treating tumors.

[0012] By deleting the TK and B13 genes of vaccinia virus and simultaneously expressing GSDME, GSDME is delivered to tumor cells and pyroptosis is induced. IL-12 is then expressed on this vector. By inducing pyroptosis (including the release of inflammatory factors such as IL-18) and IL-12 expression, the anti-tumor effect is enhanced while reducing IL-12 toxicity, achieving a synergistic anti-tumor effect of pyroptosis and IL-12. Furthermore, the vaccinia virus IL-18BP gene is simultaneously deleted from this vector to avoid the inhibition of IL-18 released by pyroptosis, thus enhancing the synergistic anti-tumor effect of IL-18 and IL-12, further strengthening the synergistic anti-tumor effect of pyroptosis (including the release of IL-18) and IL-12.

[0013] This invention provides a recombinant vaccinia virus vector that can selectively deliver GSDME to tumor cells and induce pyroptosis in them. Simultaneously, by inducing pyroptosis and co-expressing IL-12, a novel oncolytic vaccinia virus with stronger antitumor activity and higher safety is obtained. This broadens the scope of GSDME's antitumor application and will also provide a new method to improve the safety of IL-12 while maintaining its antitumor effect.

[0014] By deleting the vaccinia virus IL-18BP gene, the inhibition of IL-18 produced by pyroptosis by viral IL-18BP is avoided, further improving the antitumor efficacy and safety of recombinant vaccinia virus and providing a new perspective on the synergistic antitumor effects of pyroptosis (including its produced IL-18) and IL-12.

[0015] Given the limited anti-tumor efficacy of oncolytic viruses expressing therapeutic genes in clinical practice, and considering that GM-CSF is currently the most widely used cytokine, this invention induces pyroptosis in tumor cells while expressing GM-CSF in oncolytic vaccinia virus. This achieves the combined use of GM-CSF and the recombinant vaccinia virus vector that induces pyroptosis in tumor cells, thereby enhancing the anti-tumor efficacy of oncolytic vaccinia virus. It also provides data support for the combined use of other therapeutic genes and the recombinant vaccinia virus vector that induces pyroptosis in tumor cells of this invention.

[0016] To achieve the above objectives, the present invention adopts the following technical solution:

[0017] A method for constructing an oncolytic vaccinia virus vector involves deleting two intrinsic genes, TK and B13, of the vaccinia virus and inserting the GSDME gene. The vector induces pyroptosis in tumor cells by delivering GSDME to them, thereby killing the tumor.

[0018] The vector for the oncolytic vaccinia virus is VVΔTK-GSDMEΔB13, and the construction method includes the following steps:

[0019] (1) Using gene synthesis methods, the upstream sequence SEQ ID NO:1, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence SEQ ID NO:2, LoxP sequence, H5 promoter, human GSDME gene sequence SEQ ID NO:3 or mouse GSDME gene sequence SEQ ID NO:4 and downstream sequence SEQ ID NO:5 of the TK gene were sequentially ligated to the vector plasmid pUC57 to construct the shuttle vector plasmid pTK-GSDME; the upstream sequence SEQ ID NO:6, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence, LoxP sequence, H5 promoter and downstream sequence SEQ ID NO:7 of the B13 gene were sequentially ligated to the plasmid vector pUC57 to construct the shuttle vector plasmid pB13;

[0020] (2) Based on the TK gene sequence, design gRNA sequence SEQ ID NO:8, and ligate the gRNA sequence into the PB-gRNA vector to construct the vector plasmid PB-gRNA-TK; based on the B13 gene, design gRNA sequence SEQ ID NO:9, and ligate the gRNA sequence into the PB-gRNA vector to construct the vector plasmid PB-gRNA-B13;

[0021] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the vector plasmids Cas9 and PB-gRNA-TK. After 24 hours, the cells were infected with wild-type vaccinia virus VV. After 2 hours, the cells were transfected with the shuttle vector plasmid pTK-GSDME. After 48 hours, the mixture of supernatant and cells was collected, and the mixture was frozen and thawed once. 1 μL / well was added to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. After freezing and thawing the monoclonal solution, 5 μL / well was added to a six-well plate filled with CV1 cells. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. This was the viral vector VVΔTK-GSDME-RFP.

[0022] (4) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDME-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDME was obtained.

[0023] (5) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. After 2 hours, the cells were transfected with the shuttle vector plasmid pB13. After another 48 hours, the mixture of supernatant and cells was collected, and the mixture was frozen and thawed once. 1 μL / well was added to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. After freezing and thawing the monoclonal solution, 5 μL / well was added to a six-well plate filled with CV1 cells. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13 was obtained.

[0024] The vaccinia virus is the WR strain of vaccinia virus, with the IL-12 gene inserted into the viral vector VVΔTK-GSDMEΔB13. This induces IL-12 expression in tumor cells while simultaneously inducing pyroptosis, thus obtaining the viral vector.

[0025] The vector for the oncolytic vaccinia virus is VVΔTK-GSDMEΔB13-IL-12, and the construction method includes the following steps:

[0026] (1) Using gene synthesis methods, the upstream sequence of the B13 gene, the LoxP sequence, the H5 promoter, the reporter gene red fluorescent protein gene sequence, the LoxP sequence, the H5 promoter, the human IL-12 gene sequence SEQ ID NO:10 or the mouse IL-12 gene sequence SEQ ID NO:11 and the downstream sequence of the B13 gene are sequentially linked to the vector plasmid pUC57 to construct the shuttle vector plasmid pB13-IL12;

[0027] (2) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. After 2 hours, the cells were transfected with the shuttle vector plasmid pB13-IL12. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12-RFP was obtained.

[0028] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL12-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, they were the viral vector VVΔTK-GSDMEΔB13-IL12.

[0029] The vaccinia virus is the WR strain vaccinia virus, obtained by deleting the IL-18BP gene from the VVΔTK-GSDMEΔB13-IL12 vector.

[0030] The vector for the oncolytic vaccinia virus is VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP, and the construction method includes the following steps:

[0031] (1) The upstream sequence SEQ ID NO:12, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence, LoxP sequence, H5 promoter and downstream sequence SEQ ID NO:13 of IL-18BP gene were sequentially linked to the vector plasmid pUC57 using gene synthesis method to construct shuttle vector plasmid pIL-18BP;

[0032] (2) Based on the IL-18BP gene sequence, a gRNA sequence SEQ ID NO:14 was designed, and the gRNA sequence was ligated into the PB-gRNA vector to construct the vector plasmid PB-gRNA-IL-18BP;

[0033] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-IL-18BP vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDMEΔB13-IL-12. After 2 hours, the cells were transfected with the shuttle vector plasmid pIL-18BP. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP-RFP was obtained.

[0034] (4) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP was obtained.

[0035] The vaccinia virus is the WR strain of vaccinia virus, obtained by inserting the GM-CSF gene into the VVΔTK-GSDMEΔB13 vector.

[0036] The oncolytic vaccinia virus vector is VVΔTK-GSDMEΔB13-GMCSF, and its construction method includes the following steps:

[0037] (1) Using gene synthesis methods, the upstream sequence of the B13 gene, the LoxP sequence, the H5 promoter, the RFP gene sequence, the LoxP sequence, the H5 promoter, the human GM-CSF gene sequence SEQ ID NO:15 or the mouse GM-CSF gene sequence SEQ ID NO:16 and the downstream sequence of the B13 gene are sequentially linked to the vector plasmid pUC57 to construct the shuttle vector plasmid pB13-GMCSF;

[0038] (2) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. After 2 hours, the cells were transfected with the shuttle vector plasmid pB13-GM CSF. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF-RFP was obtained.

[0039] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-GMCSF-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-GMCSF was obtained.

[0040] The oncolytic vaccinia virus vector obtained by the method is used in the preparation of drugs for treating tumors or infectious diseases.

[0041] The tumors mentioned are solid tumors, and the infectious diseases are viral, bacterial, or fungal infections. Solid tumors include pancreatic cancer, lung cancer, ovarian cancer, colorectal cancer, esophageal cancer, liver cancer, cervical cancer, melanoma, glioma, breast cancer, and kidney cancer.

[0042] The present invention discloses a method for constructing a tumor-targeting oncolytic vaccinia virus vector by deleting two intrinsic genes, the TK gene and the B1 gene 3, of the virus and inserting the GSDME gene into the TK region, thereby constructing an oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13 that selectively delivers GSDME to tumor cells and induces pyroptosis. This vector can also be inserted with any gene that may be helpful in treating tumors or used in vaccines for infectious diseases.

[0043] This invention inserts the IL-12 gene into the vector VVΔTK-GSDMEΔB13, thereby constructing an oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 that induces pyroptosis and expresses IL-12. Compared with the control virus vector VVΔTKΔB13-IL12 that only expresses IL-12, this vector can significantly reduce IL-12 toxicity and exert a better anti-tumor effect.

[0044] To prevent the IL-18 produced by pyroptosis from being bound by the viral IL-18BP, the IL-18BP gene was subsequently deleted, thereby constructing the VVΔTK-GSDMEΔB13-IL12ΔIL-18BP recombinant vaccinia virus vector, which can further improve the anti-tumor efficacy.

[0045] By inserting the GM-CSF gene into the tumor-targeting viral vector VVΔTK-GSDMEΔB13, a recombinant vector VVΔTK-GSDMEΔB13-GMCSF was constructed, enabling the combined use of GM-CSF and the pyroptosis-inducing vaccinia virus vector to further enhance the antitumor effect of the recombinant vaccinia virus.

[0046] The recombinant vectors VVΔTK-GSDMEΔB13-IL12 and VVΔTK-GSDMEΔB13-IL12ΔIL-18BP of this invention can selectively replicate in tumor cells and express GSDME to induce pyroptosis and IL-12 after entering the tumor. As tumor cells undergo pyroptosis and lysis, inflammatory substances, tumor-associated antigens, and viral particles are released. The viral particles can continue to infect nearby tumor cells. The released inflammatory substances and tumor-associated antigens work synergistically with IL-12 to induce a highly efficient and specific anti-tumor immune response in the body, further killing tumor cells that have not been infected by the virus, including distant tumors or tumor metastases.

[0047] VVΔTK-GSDMEΔB13 successfully induced pyroptosis in tumor cells while exhibiting good safety. VVΔTK-GSDMEΔB13-IL12 induced pyroptosis in tumor cells while reducing IL-12 secretion levels. This recombinant vector can sustainably and stably express IL-12 at relatively low levels, thus compensating for the shortcomings of existing recombinant IL-12 proteins, such as short half-life and the toxicity caused by high-level expression. Furthermore, IL-12 and IL-18 work synergistically; pyroptosis produces IL-18. To avoid the inhibitory effect of virally produced IL-18BP on pyroptosis-produced IL-18, the VVΔTK-GSDMEΔB13-IL12ΔIL-18BP recombinant vector deleted the viral IL-18BP gene, resulting in a stronger anti-tumor efficacy. VVΔTK-GSDMEΔB13-GMCSF expressed GM-CSF while inducing pyroptosis in tumor cells, enhancing the anti-tumor efficacy of recombinant vaccinia virus.

[0048] The beneficial effects of this invention are:

[0049] (1) The tumor-targeting viral vector VVΔTK-GSDMEΔB13 constructed in this invention deletes the viral TK gene and B13 gene while expressing GSDME, resulting in a safe and effective method for delivering GSDME to tumor cells and inducing pyroptosis of tumor cells, showing better tumor suppression effect and improving the survival of tumor-bearing mice.

[0050] (2) The VVΔTK-GSDMEΔB13-IL12 recombinant vector of the present invention expresses IL-12 while inducing pyroptosis in tumor cells, achieving sustained and stable IL-12 expression at a relatively low level. This significantly reduces the toxicity of IL-12 while maintaining its excellent anti-tumor effect, thus solving the problems of IL-12 recombinant protein toxicity and short half-life. Furthermore, the deletion of the viral IL-18BP gene avoids the inhibitory effect of viral IL-18BP on IL-18 production during pyroptosis, further improving the safety and efficacy of treatment. This demonstrates the superior anti-tumor effect of the VVΔTK-GSDMEΔB13-IL12ΔIL-18BP recombinant vector in treating subcutaneous xenograft tumors in mice.

[0051] (3) The oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 of the present invention can be used not only for intratumoral injection but also for intravenous injection. Both treatment methods have shown good safety. Intravenous injection is an ideal systemic drug delivery method, which reduces the difficulty of drug delivery and expands the scope of tumor treatment. It is applicable to non-surface tumors, multiple tumors and metastatic tumors, etc., and meets the clinical needs of most tumor treatments.

[0052] (4) The tumor-targeting viral vector VVΔTK-GSDMEΔB13-GMCSF of the present invention expresses GSDME and GM-CSF genes while deleting the viral B13 gene. It achieves a significant improvement in anti-tumor efficacy by combining pyroptosis with the most widely used cytokine GM-CSF, providing data support and new perspectives for the combined use of pyroptosis and more therapeutic genes, and has good application prospects. Attached Figure Description

[0053] Figure 1. Schematic diagram of the structures of recombinant vaccinia virus vectors VVΔTK-GSDMEΔB13, VVΔTK-GSDMEΔB13-IL12, VVΔTK-GSDMEΔB13-IL12ΔIL-18BP, VVΔTK-GSDMEΔB13-GMCSF and control virus.

[0054] Figure 2. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13 and control virus on common mouse tumor cells.

[0055] Figure 3. The therapeutic effects and survival curves of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13 and control virus on a mouse subcutaneous breast cancer tumor model.

[0056] Figure 4. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and control virus on common mouse tumor cells.

[0057] Figure 5. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and control virus on common human tumor cells.

[0058] Figure 6. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and control virus on hamster tumor cells.

[0059] Figure 7. Comparison of the IL-12 expression capabilities of the recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and the control virus.

[0060] Figure 8. The therapeutic effects and weight changes of the recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and the control virus on a mouse subcutaneous breast cancer tumor model.

[0061] Figure 9. The therapeutic effects and weight changes of the recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and the control virus on a mouse model of subcutaneous pancreatic cancer.

[0062] Figure 10 Survival curves and tumor clearance rates of mouse pancreatic cancer subcutaneous tumor models treated with recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 and control virus.

[0063] Figure 11. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12ΔIL-18BP and control virus on common mouse tumor cells.

[0064] Figure 12. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12ΔIL-18BP and control virus on common human tumor cells.

[0065] Figure 13. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12ΔIL-18BP and control virus on hamster tumor cells.

[0066] Figure 14. The therapeutic effects and tumor clearance rate of the recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-IL12ΔIL-18BP and control virus on a mouse subcutaneous colorectal cancer model.

[0067] Figure 15. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF and control virus on common mouse tumor cells.

[0068] Figure 16. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF and control virus on common human tumor cells.

[0069] Figure 17. Killing effect of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF and control virus on hamster tumor cells.

[0070] Figure 18. The therapeutic effect and tumor clearance rate of recombinant vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF and control virus on mouse melanoma subcutaneous tumor model. Detailed Implementation

[0071] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0072] The tumor-targeting vaccinia virus vector in this embodiment of the invention is the Western Reserve (WR) strain, but other vaccinia virus strains such as the Copenhagen strain, Lister strain, Wyeth strain, or Tiantan strain can also be used.

[0073] Example 1. Construction of tumor-targeting oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13

[0074] (1) First, gene fragments flanking the TK gene and B13 gene, and the reporter gene red fluorescent protein (RFP) gene sequence were obtained using gene synthesis methods. The upstream sequences of the TK and B13 genes are referred to as the left arm, and the downstream sequences as the right arm. The upstream sequences of the TK gene (SEQ ID NO:1), LoxP sequence, H5 promoter, RFP sequence (SEQ ID NO:2), LoxP sequence, H5 promoter, GSDME gene sequence (human SEQ ID NO:3, mouse SEQ ID NO:4, human gene prefixed with h, mouse gene prefixed with m) and the downstream sequence of the TK gene (SEQ ID NO:5) were sequentially ligated into the vector plasmid pUC57 to construct the shuttle vector plasmid pTK-GSDME. The upstream sequences of the B13 gene (SEQ ID NO:6), LoxP sequence, H5 promoter, RFP gene sequence, LoxP sequence, H5 promoter, and the downstream sequence of the B13 gene (SEQ ID NO:6) were sequentially ligated into the vector plasmid pUC57 to construct the shuttle vector plasmid pTK-GSDME. NO:7 was linked to the vector plasmid pUC57 to construct the shuttle vector plasmid pB13.

[0075] (2) Based on the TK gene sequence, design gRNA sequence SEQ ID NO:8, and ligate the gRNA sequence into the PB-gRNA vector to construct the vector plasmid PB-gRNA-TK; based on the B13 gene, design gRNA sequence SEQ ID NO:9, and ligate the gRNA sequence into the PB-gRNA vector to construct the vector plasmid PB-gRNA-B13;

[0076] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the vector plasmids Cas9 and PB-gRNA-TK. After 24 hours, the cells were infected with wild-type vaccinia virus VV. After 2 hours, the cells were transfected with the shuttle vector plasmid pTK-GSDME. After 48 hours, the mixture of supernatant and cells was collected, and the mixture was frozen and thawed once. 1 μL / well was added to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. After freezing and thawing the monoclonal solution, 5 μL / well was added to a six-well plate filled with CV1 cells. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. This was the viral vector VVΔTK-GSDME-RFP.

[0077] (4) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDME-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDME was obtained.

[0078] (5) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDME. After 2 hours, they were transfected with the shuttle vector plasmid pB13. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13 was obtained (see Figure 1 for the structural diagram).

[0079] The control viral vectors VVΔTK, VVΔTKΔB13, and VVΔTK-GSDME were constructed using the same method (see Figure 1).

[0080] Example 2. Construction of tumor-targeted oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12

[0081] (1) The upstream sequence of the B13 gene, the LoxP sequence, the H5 promoter, the RFP, the LoxP sequence, the H5 promoter, the IL-12 gene sequence (human SEQ ID NO:10, mouse SEQ ID NO:11) and the downstream sequence of the B13 gene were sequentially linked to the plasmid vector pUC57 to construct the shuttle vector plasmid pB13-IL12.

[0082] (2) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDME. After 2 hours, they were transfected with the shuttle vector plasmid pB13-IL12. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added to a six-well plate filled with CV1 cells at a rate of 1 μL / well. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added to a six-well plate filled with CV1 cells at a rate of 5 μL / well. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12-RFP was obtained.

[0083] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL12-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-IL12 (abbreviated as VV-DD-GE12, DD, double deletion, refers to the deletion of the TK gene and the B13 gene) was obtained (see Figure 1 for the structural diagram).

[0084] The control viral vector VVΔTKΔB13-IL12 was constructed using the same method.

[0085] Example 3. Construction of tumor-targeted oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP

[0086] (1) The upstream sequence SEQ ID NO:12, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence, LoxP sequence, H5 promoter and downstream sequence SEQ ID NO:13 of IL-18BP gene were sequentially linked to the vector plasmid pUC57 using gene synthesis method to construct shuttle vector plasmid pIL-18BP.

[0087] (2) Based on the IL-18BP gene sequence, a gRNA sequence SEQ ID NO:14 was designed and the gRNA sequence was ligated into the PB-gRNA vector to construct the vector plasmid PB-gRNA-IL-18BP.

[0088] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-IL-18BP vector plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL-12. After 2 hours, they were transfected with the shuttle vector plasmid pIL-18BP. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added to a six-well plate filled with CV1 cells at 1 μL / well. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. After freezing and thawing the monoclonal solution, it was added to a six-well plate filled with CV1 cells at 5 μL / well. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP-RFP was obtained.

[0089] (4) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP-RFP. After another 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP (abbreviated as VV-TD-GE12, TD stands for triple deletion, which means the deletion of TK gene, B13 gene and IL-18BP gene) was obtained (see Figure 1 for the structural diagram).

[0090] The control viral vector VVΔTK-GSDMEΔB13ΔIL-18BP (VV-TD-GE) was constructed using the same method.

[0091] Example 4. Construction of tumor-targeted oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF

[0092] (1) The upstream sequence of the B13 gene, the LoxP sequence, the H5 promoter, the RFP gene sequence, the LoxP sequence, the H5 promoter, the GM-CSF gene sequence (human SEQ ID NO:15, mouse SEQ ID NO:16) and the downstream sequence of the B13 gene were sequentially linked to the vector plasmid pUC57 to construct the shuttle vector plasmid pB13-GMCSF.

[0093] (2) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. Two hours later, the cells were transfected with the shuttle vector plasmid pB13-GMCSF. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF-RFP was obtained.

[0094] (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-GMCSF-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-GMCSF (abbreviated as VV-DD-GEGF) was obtained (see Figure 1 for the structural diagram).

[0095] The control viral vector VVΔTKΔB13-GMCSF was constructed using the same method.

[0096] Example 5. Detection of the killing effects of four tumor-targeting oncolytic vaccinia virus vectors VVΔTK-GSDMEΔB13, VVΔTK-GSDMEΔB13-IL12, VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP, and VVΔTK-GSDMEΔB13-GMCSF on human, mouse, and hamster tumor cells.

[0097] The LDH method was used to detect the killing ability of recombinant vaccinia virus against tumor cells. The amount of LDH in the supernatant was used to reflect the in vitro killing ability of recombinant vaccinia virus against tumor cells. The higher the LDH level, the stronger the killing ability of recombinant vaccinia virus against tumor cells.

[0098] The tumor cell lines involved include: seven mouse tumor cell lines, including mouse breast cancer cells 4T1, mouse melanoma cells B16-F10, mouse pancreatic cancer cells DT6606, mouse pancreatic cancer cells TB11381, mouse colorectal cancer cells MC38, mouse colorectal cancer cells CT26, and mouse glioma cells GL261; seven human tumor cell lines, including human ovarian cancer cells SKOV3, human liver cancer cells HepG2, human cervical cancer cells HeLa, human colorectal cancer cells HCT116, human lung cancer cells A549, human pancreatic cancer cells SUIT2, and human esophageal cancer cells KYSE150; and two hamster tumor cell lines, including hamster pancreatic cancer cells HPD-1NR and hamster kidney cancer cells HaK.

[0099] (1) Mouse cancer cells 4T1, B16-F10, DT6606, GL261, CT26, MC38, and TB11381 in logarithmic growth phase were collected, counted, and seeded in 96-well plates at 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTK, VVΔTKΔB13 (VV-DD), VVΔTK-mGSDME (VVΔTK-mGE), and VVΔTK-mGSDMEΔB13 (VV-DD-mGE). A blank control (Mock) was also set up (without virus). The supernatant was collected 36-72 hours after virus infection, and LDH (lactate dehydrogenase, Promega's LDH-Glo™ Cytotoxicity Assay kit) was detected and calculated. See Figure 2.

[0100] As shown in Figure 2, VV-DD-mGE exhibits a stronger killing effect on common mouse tumor cells compared to the control virus. In the figure, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001 (the asterisks in other figures have the same meaning).

[0101] (2) Mouse cancer cells 4T1, B16-F10, DT6606, GL261, MC38, CT26, and TB11381 in logarithmic growth phase were collected, counted, and seeded in 96-well plates at 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTKΔB13-mIL12 (VV-DD-mIL12) and VVΔTK-mGSDMEΔB13-mIL12 (VV-DD-mGE12), respectively. A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated. As shown in Figure 4, VV-DD-mGE12 had a stronger killing effect on common mouse tumor cells compared with the control virus.

[0102] (3) Human cancer cells in the logarithmic growth phase, including HeLa, HCT116, HepG2, SKOV3, SUIT2, KYSE150, and A549, were collected, counted, and seeded in 96-well plates at a rate of 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTK-hGSDMEΔB13 (VV-DD-hGE), VVΔTKΔB13-hIL12 (VV-DD-hIL12), and VVΔTK-hGSDMEΔB13-hIL12 (VV-DD-hGE12). A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated.

[0103] As shown in Figure 5, VV-DD-hGE and VV-DD-hGE12 have stronger killing effects on common human tumor cells compared with VV-DD-hIL12.

[0104] (4) Syrian hamster cancer cells HPD-1NR and HaK in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTK-hGSDMEΔB13 (VV-DD-hGE), VVΔTKΔB13-hIL12 (VV-DD-hIL12), and VVΔTK-hGSDMEΔB13-hIL12 (VV-DD-hGE12). A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated. As shown in Figure 6, VV-DD-hGE and VV-DD-hGE12 had stronger killing effects on hamster tumor cells compared with VV-DD-hIL12.

[0105] (5) Mouse cancer cells 4T1, B16-F10, DT6606, MC38, and CT26 in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at a rate of 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTKmGSDMEΔB13-mIL12 (VV-DD-mGE12) and VVΔTK-mGSDMEΔB13-mIL12-ΔIL-18BP (VV-TD-mGE12), respectively. A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated.

[0106] As shown in Figure 11, VV-DD-mGE12 and VV-TD-mGE12 have similar anti-tumor effects, and deletion of the IL-18BP gene does not affect the killing effect of recombinant vaccinia virus on common mouse tumor cells.

[0107] (6) Human cancer cells in the logarithmic growth phase, namely HeLa, A549, SKOV3, HCT116, HepG2, KYSE150, and SUI T2, were collected, counted, and seeded in 96-well plates at a rate of 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The viruses added were VVΔTK-hGSDMEΔB13 (VV-DD-hGE), VVΔTK-hGSDM EΔB13-ΔIL-18BP (VV-TD-hGE), VVΔTK-hGSDMEΔB13-hIL12 (VV-DD-hGE12), and VVΔTK-hGSDMEΔB13-hIL12-ΔIL-18BP (VV-TD-hGE12). A blank control mock was also set up. The supernatant was collected 36-72 hours after viral infection, and LDH was detected and calculated.

[0108] As shown in Figure 12, deletion of the IL-18BP gene did not reduce the killing effect of recombinant vaccinia virus on common human tumor cells, while VV-TD-hGE and VV-TD-hGE12 enhanced the killing effect on HeLa, HCT116 and HepG2 cells.

[0109] (7) Syrian hamster cancer cells HPD-1NR and HaK in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTK-hGSDMEΔB13 (VV-DD-hGE), VVΔTK-hGSDMEΔB13-ΔIL-18BP (VV-TD-hGE), VVΔTK-hGSDMEΔB13-hIL12 (VV-DD-hGE12), and VVΔTK-hGSDMEΔB13-hIL12-ΔIL-18BP (VV-TD-hGE12). A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated.

[0110] As shown in Figure 13, deletion of the IL-18BP gene did not reduce the killing effect of recombinant vaccinia virus on hamster tumor cells, while VV-TD-hGE and VV-TD-hGE12 enhanced the killing effect on HPD1NR cells.

[0111] (8) Mouse cancer cells 4T1, B16-F10, DT6606, and MC38 in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at a rate of 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTKΔB13-mGMCSF (VV-DD-mGF) and VVΔTK-mGSDMEΔB13-mGMCSF (VV-DD-mGEGF), respectively. A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated.

[0112] As shown in Figure 15, VV-DD-mGEGF has a stronger killing effect on common mouse tumor cells compared with VV-DD-mGE.

[0113] (9) Human cancer cells A549, SKOV3, HCT116, HepG2, SUIT2, HeLa, and KYSE 150 in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTKΔB13-hGMCSF (VV-DD-hGF), VVΔTK-hGSDMEΔB13 (VV-DD-hGE), and VVΔTK-hGSDMEΔB13-hGMCSF (VV-DD-hGEGF). A blank control mock was also set up. The supernatant was collected 36-72 hours after viral infection, and LDH was detected and calculated.

[0114] As shown in Figure 16, VV-DD-hGE and VV-DD-hGEGF have stronger killing effects on human A549, HCT116, HepG2 and SUIT2 tumor cells compared with VV-DD-hGF.

[0115] (10) Syrian hamster cancer cells HPD1NR and HaK in the logarithmic growth phase were collected, counted, and seeded in 96-well plates at 1×10^4 cells / well. After observing cell adhesion for 12-16 hours, the virus was diluted and added to the 96-well plates at an MOI of 10. The added viruses were VVΔTKΔB13-hGMCSF (VV-DD-hGF), VVΔTK-hGSDMEΔB13 (VV-DD-hGE), and VVΔTK-hGSDMEΔB13-hGMCSF (VV-DD-hGEGF). A blank control mock was also set up. The supernatant was collected 36-72 hours after virus infection, and LDH was detected and calculated.

[0116] As shown in Figure 17, VV-DD-hGEGF has a stronger killing effect on hamster HaK tumor cells compared with VV-DD-hGF.

[0117] Example 6. Detection of the ability of tumor-targeted oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 to express IL-12

[0118] Mouse breast cancer 4T1 cells were infected with VVΔTK-mGSDMEΔB13-mIL12 (VV-DD-mGE12) and control virus VVΔTKΔB13-mIL12 (VV-DD-mIL12) at an MOI of 10. A blank control Mock was also set up. The supernatant was collected at 12h, 24h, 36h, 48h and 60h after infection, and the expression of IL-12 after virus infection was detected by ELISA.

[0119] As shown in Figure 7, both VV-DD-mGE12 and the control virus VV-DD-mIL12 were able to successfully express and secrete IL-12 after infecting tumor cells. Compared with the control virus VV-DD-mIL12, VV-DD-mGE12 could significantly reduce the expression of IL-12 (ND indicates that no IL-12 expression was detected).

[0120] Example 7. Detection of the therapeutic effect of tumor-targeted oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13 on a mouse subcutaneous breast cancer tumor model.

[0121] BALB / c female mice were subcutaneously injected with 5×10⁵ cells on the right back. 5 A mouse breast cancer 4T1 cell was observed, and the tumor volume was approximately 60 mm after 5 days. 3 Mice were randomly divided into 5 groups of 7 each. On days 0, 2, and 4, the tumors were injected with either PBS or 2×10⁻⁶ ppm of PBS. 8 PFU containing recombinant vaccinia virus VVΔTK, VV-DD), VVΔTK-mGSDME (VVΔTK-mGE), and VVΔTK-mGSDMEΔB13 (VV-DD-mGE) was used to measure tumor volume every other day.

[0122] As shown in Figure 3, the tumor growth curve of the VV-DD-mGE treatment group increased the slowest after treatment, which inhibited tumor growth and showed the strongest anti-tumor ability; the mice had the longest survival time, which significantly prolonged the survival period of the mice in the treatment group.

[0123] Example 8: Detection of the therapeutic effect of tumor-targeting vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 on a mouse subcutaneous breast cancer tumor model.

[0124] BALB / c female mice were subcutaneously injected with 5×10⁵ cells on the right back. 5 A mouse breast cancer 4T1 cell was observed, and the tumor volume was approximately 120 mm² after 7 days. 3 Mice were randomly divided into three groups of seven each. PBS or 5×10⁻⁶ ppm precipitates were injected into the tumor on days 0, 2, 4, and 6. 7 PFU-containing recombinant vaccinia virus VVΔTKΔB13-mIL12 (VV-DD-mIL12) and VVΔTK-mGSDMEΔB13-mIL12 (VV-DD-mGE12) were used to measure tumor volume and mouse body weight every other day.

[0125] As shown in Figure 8, both VV-DD-mIL12 and VV-DD-mGE12 exhibited superior antitumor activity, with no statistically significant difference between the two groups. The change in body weight indicates that VV-DD-mGE12 significantly reduced treatment toxicity compared to VV-DD-mIL12. These results suggest that intratumoral injection of VVΔTK-VV-DD-mGE12 demonstrates excellent antitumor activity while maintaining a good safety profile.

[0126] Example 9. Detection of the therapeutic effect of tumor-targeting vaccinia virus vector VVΔTK-GSDMEΔB13-IL12 on a mouse model of subcutaneous pancreatic cancer.

[0127] C57BL / 6N male mice were subcutaneously injected with 2×10⁻⁶ mice on the right back. 8 A mouse pancreatic cancer DT6606 cell line was used, and after 7 days, the tumor volume was approximately 120 mm. 3 Mice were randomly divided into 4 groups of 7 each. On days 0 and 2, they were injected via the tail vein with PBS or 3×10⁻⁶ ppm PBS. 7 PFU-containing recombinant vaccinia virus VVΔTK-mGSDMEΔB13 (VV-DD-mGE), VVΔTKΔB13-mIL12 (VV-DD-mIL12), and VVΔTK-mGSDMEΔB13-mIL12 (VV-DD-mGE12) were used to measure tumor volume and mouse body weight every other day.

[0128] As shown in Figure 9, intravenous injection of VV-DD-mGE can effectively inhibit tumor growth; compared with VV-DD-mIL12, VV-DD-mGE12 exhibits stronger anti-tumor efficacy and better safety.

[0129] As shown in Figure 10, intravenous injection of VV-DD-mGE effectively prolonged the survival of mice; compared with VV-DD-mIL12, VV-DD-mGE12 significantly prolonged the survival of mice and completely cleared 42.9% of the tumors in the mice. These results indicate that intravenous injection of VV-DD-mGE12 exhibits excellent anti-tumor efficacy while maintaining good safety.

[0130] Example 10. Detection of the therapeutic effect of tumor-targeted vaccinia virus vector VVΔTK-GSDMEΔB13-IL12ΔIL-18BP on a mouse subcutaneous colon cancer model.

[0131] C57BL / 6N female mice were subcutaneously injected with 1×10⁻⁶ cells on the right back. 8 A mouse colon cancer MC38 cell line was used, and after 10 days, the tumor volume was approximately 200 mm. 3 Mice were randomly divided into three groups of six each. On days 0, 2, and 4, the tumors were injected with PBS or 5×10⁻⁶ ppm PBS. 7PFU-containing recombinant vaccinia virus VVΔTK-mGSDMEΔB13-mIL12 (VV-DD-mGE12) and VVΔTK-mGSDMEΔB13-mIL 12ΔIL-18BP (VV-TD-mGE12) were used to measure tumor volume and mouse weight every 2-3 days.

[0132] As shown in Figure 14, compared with VV-DD-mGE12, VV-TD-mGE12 exhibited stronger anti-tumor ability and higher tumor clearance rate, clearing all tumors in mice, while VV-DD-mGE12 only cleared 33.3% of mouse tumors.

[0133] Example 11. Detection of the therapeutic effect of tumor-targeting vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF on a mouse subcutaneous melanoma model.

[0134] C57BL / 6N female mice were subcutaneously injected with 5×10⁻⁶ cells on the right back. 5 A mouse melanoma B16-F10 cell was observed, and the tumor volume was approximately 70 mm² after 6 days. 3 Mice were randomly divided into three groups of eight each. On days 0, 2, and 4, the tumors were injected with either PBS or 2×10⁻⁶ ppm of PBS. 8 PFU containing recombinant vaccinia virus VVΔTKΔB13-mGMCSF (VV-DD-mGF) and VVΔTK-mGSDMEΔB13-mGMCSF (VV-DD-mGEGF), with tumor volume measured every 1-2 days.

[0135] As shown in Figure 18, compared with VV-DD-mGF, VV-DD-mGEGF exhibited stronger anti-tumor activity and a higher tumor clearance rate, clearing 62.5% of mouse tumors, while VV-DD-mGF could not clear mouse tumors.

[0136] Table 1. Full names and abbreviations of viruses

[0137] Note: Adding "m" before the abbreviation indicates mouse-derived genes, and adding "h" indicates human-derived genes.

Claims

1. A method of constructing an oncolytic vaccinia virus vector, characterized by, The TK and B13 genes of vaccinia virus were deleted, and the GSDME gene was inserted. By delivering GSDME to tumor cells, pyroptosis was induced, thereby killing tumor cells.

2. The method of constructing an oncolytic vaccinia virus vector according to claim 1, wherein, The vector for the oncolytic vaccinia virus is VVΔTK-GSDMEΔB13, and the construction method includes the following steps: (1) Using gene synthesis methods, the upstream sequence SEQ ID NO:1, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence SEQ ID NO:2, LoxP sequence, H5 promoter, GSDME gene sequence and the downstream sequence SEQ ID NO:5 of the TK gene were sequentially ligated to the vector plasmid pUC57 to construct the shuttle vector plasmid pTK-GSDME; the upstream sequence SEQ ID NO:6, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence, LoxP sequence, H5 promoter and the downstream sequence SEQ ID NO:7 of the B13 gene were sequentially ligated to the plasmid vector pUC57 to construct the shuttle vector plasmid pB13; (2) Based on the TK gene sequence, design gRNA sequence SEQ ID NO:8, and ligate the gRNA sequence into the PB-gRNA vector to construct the vector plasmid PB-gRNA-TK; based on the B13 gene, design gRNA sequence SEQ ID NO:9, and ligate the gRNA sequence into the PB-gRNA vector to construct the vector plasmid PB-gRNA-B13; (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the vector plasmids Cas9 and PB-gRNA-TK. After 24 hours, the cells were infected with wild-type vaccinia virus VV. After 2 hours, the cells were transfected with the shuttle vector plasmid pTK-GSDME. After 48 hours, the mixture of supernatant and cells was collected, and the mixture was frozen and thawed once. 1 μL / well was added to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. After freezing and thawing the monoclonal solution, 5 μL / well was added to a six-well plate filled with CV1 cells. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. This was the viral vector VVΔTK-GSDME-RFP. (4) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDME-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDME was obtained. (5) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. After 2 hours, the cells were transfected with the shuttle vector plasmid pB13. After another 48 hours, the mixture of supernatant and cells was collected, and the mixture was frozen and thawed once. 1 μL / well was added to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. After freezing and thawing the monoclonal solution, 5 μL / well was added to a six-well plate filled with CV1 cells. Monoclonal cells were picked again after 48 hours until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13 was obtained.

3. A method of constructing an oncolytic vaccinia virus vector as claimed in claim 2, characterized in that, The vaccinia virus is the WR strain of vaccinia virus, with the IL-12 gene inserted into the viral vector VVΔTK-GSDMEΔB13. This induces IL-12 expression in tumor cells while simultaneously inducing pyroptosis, thus obtaining the viral vector.

4. The method of constructing an oncolytic vaccinia virus vector according to claim 3, wherein, The vector for the oncolytic vaccinia virus is VVΔTK-GSDMEΔB13-IL-12, and the construction method includes the following steps: (1) The upstream sequence of the B13 gene, the LoxP sequence, the H5 promoter, the reporter gene red fluorescent protein gene sequence, the LoxP sequence, the H5 promoter, the IL-12 gene sequence and the downstream sequence of the B13 gene were sequentially linked to the vector plasmid pUC57 to construct the shuttle vector plasmid pB13-IL12. (2) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. After 2 hours, the cells were transfected with the shuttle vector plasmid pB13-IL12. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL12-RFP was obtained. (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL12-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, they were the viral vector VVΔTK-GSDMEΔB13-IL12.

5. A method of constructing an oncolytic vaccinia virus vector according to claim 4, wherein, The vaccinia virus is the WR strain of vaccinia virus, obtained by deleting the IL-18BP gene from the VVΔTK-GSDMEΔB13-IL12 vector.

6. The method of constructing an oncolytic vaccinia virus vector according to claim 5, wherein, The vector for the oncolytic vaccinia virus is VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP, and the construction method includes the following steps: (1) The upstream sequence SEQ ID NO:12, LoxP sequence, H5 promoter, reporter gene red fluorescent protein gene sequence, LoxP sequence, H5 promoter and downstream sequence SEQ ID NO:13 of IL-18BP gene were sequentially linked to the vector plasmid pUC57 using gene synthesis method to construct shuttle vector plasmid pIL-18BP; (2) Based on the IL-18BP gene sequence, a gRNA sequence SEQ ID NO:14 was designed, and the gRNA sequence was ligated into the PB-gRNA vector to construct the vector plasmid PB-gRNA-IL-18BP; (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-IL-18BP vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDMEΔB13-IL-12. After 2 hours, the cells were transfected with the shuttle vector plasmid pIL-18BP. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP-RFP was obtained. (4) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-IL-12ΔIL-18BP was obtained.

7. A method of constructing an oncolytic vaccinia virus vector as claimed in claim 2, wherein, The vaccinia virus is the WR strain of vaccinia virus, obtained by inserting the GM-CSF gene into the VVΔTK-GSDMEΔB13 vector.

8. The method of constructing an oncolytic vaccinia virus vector according to claim 7, wherein, The oncolytic vaccinia virus vector is VVΔTK-GSDMEΔB13-GMCSF, and its construction method includes the following steps: (1) The upstream sequence of the B13 gene, the LoxP sequence, the H5 promoter, the RFP gene sequence, the LoxP sequence, the H5 promoter, the GM-CSF gene sequence and the downstream sequence of the B13 gene were sequentially linked to the vector plasmid pUC57 to construct the shuttle vector plasmid pB13-GMCSF. (2) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were simultaneously transfected with the Cas9 vector plasmid and the PB-gRNA-B13 vector plasmid. After 24 hours, the cells were infected with the viral vector VVΔTK-GSDME. After 2 hours, the cells were transfected with the shuttle vector plasmid pB13-GMCSF. After another 48 hours, the mixture of supernatant and cells was collected, frozen and thawed once, and added at 1 μL / well to a six-well plate filled with CV1 cells. After 48 hours, red fluorescent monoclonal cells were picked under a fluorescence microscope. The monoclonal solution was frozen and thawed and added at 5 μL / well to a six-well plate filled with CV1 cells. After 48 hours, monoclonal cells were picked again until all cells were red fluorescent under a fluorescence microscope. The recombinant oncolytic vaccinia virus vector VVΔTK-GSDMEΔB13-GMCSF-RFP was obtained. (3) CV1 cells were seeded into six-well plates. When the cells reached a confluence of more than 90%, they were transfected with Cre plasmid. After 24 hours, they were infected with the viral vector VVΔTK-GSDMEΔB13-GMCSF-RFP. After 48 hours, single clones without red fluorescence were picked. After multiple rounds of selection, until all cells were without fluorescence under a fluorescence microscope, the viral vector VVΔTK-GSDMEΔB13-GMCSF was obtained.

9. The use of the oncolytic vaccinia virus vector obtained by the method according to any one of claims 2, 4, 6, and 8 in the preparation of medicaments for treating tumors or infectious diseases.

10. Use according to claim 9, wherein The tumor is a solid tumor, and the infectious disease is a viral, bacterial, or fungal infection.

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