A tumor-specific pyroptosis induction system based on transcription amplification principle and application thereof in preparation of anti-tumor drugs

Abstract

The application provides a tumor-specific pyroptosis induction system based on transcription amplification principle and application thereof in preparation of an antitumor drug. The system comprises a first expression vector and a second expression vector. The first expression vector comprises a tumor-specific promoter and a site-specific recombinase gene regulated by the tumor-specific promoter. The second expression vector comprises a strong promoter, a pyroptosis induction protein gene and a transcription blocking element arranged between the strong promoter and the pyroptosis induction protein gene. The transcription blocking element is composed of two same-direction recombinant sites at the two sides and a transcription termination sequence in the middle. In a tumor cell, the tumor-specific promoter activates and expresses the recombinase, the transcription termination sequence is excised to remove the transcription block, the pyroptosis induction protein is efficiently driven to express by the strong promoter, and the tumor cell pyroptosis is specifically induced. The application significantly improves the tumor specificity and expression efficiency of pyroptosis induction, balances the treatment effectiveness and safety, and is suitable for antitumor treatment of solid tumors.

Description

Technical Field

[0001] This invention relates to a tumor-specific pyroptosis induction system based on the principle of transcriptional amplification and its application in the preparation of antitumor drugs. Background Technology

[0002] Targeted elimination of tumor cells and maximum protection of normal tissues are core objectives in the treatment of malignant tumors. Although conventional clinical therapies such as chemotherapy, radiotherapy, and immune checkpoint blockade have made some progress in the treatment of solid tumors, these methods often lead to serious systemic side effects (such as myelosuppression and immune-related pneumonia) due to significant off-target effects, and their narrow therapeutic window significantly limits their clinical application. It is worth noting that while targeted therapies (such as EGFR-TKIs) and novel immunotherapies (such as CAR-T) in the era of precision medicine have significantly improved survival benefits in some cancer types (such as non-small cell lung cancer and hematological malignancies) through molecular subtyping guidance, acquired resistance due to tumor heterogeneity (such as T790M mutations) and suppression of the immune microenvironment remain common challenges in the clinical management of solid tumors. Therefore, developing novel treatment strategies that combine high tumor-killing efficiency with low toxicity has become a key research direction for overcoming current bottlenecks in the treatment of solid tumors.

[0003] Pyroptosis is a form of programmed cell death mediated by downstream effector caspases (caspase-1 / 4 / 5 / 11) of the inflammasome, exhibiting significant immunogenicity and pro-inflammatory properties. Its molecular mechanism primarily relies on the specific cleavage of gasdermin family proteins (GSDMs) by effector caspases, releasing the N-terminal domain (GSDM) with membrane perforation activity. NTThis domain can self-assemble on the cell membrane to form 10-20 nm oligomeric channels, leading to osmotic imbalance, membrane rupture, and the release of large amounts of pro-inflammatory factors (such as IL-1β and IL-18) and damage-associated molecular patterns (DAMPs). From a pathophysiological perspective, pyroptosis can cause tissue damage in normal tissues and is closely related to diseases such as sepsis, acute lung injury, and autoimmune disorders. In the tumor microenvironment, pyroptosis can induce a strong anti-tumor immune response by releasing tumor-associated antigens (TAAs), activating antigen-presenting cells (APCs), and remodeling the immunosuppressive microenvironment. Related studies have shown that pyroptosis is positively correlated with immune infiltration and immune-related characteristics of 30 types of cancer. Inducing pyroptosis in tumor tissue is a promising cancer immunotherapy strategy; even pyroptosis in 15% of tumor cells can completely eliminate tumor tissue in mouse xenograft models. However, existing treatment strategies based on bioorthogonal chemistry, intratumoral injection, or low-dose GSDMD agonists still face significant challenges in terms of tumor specificity. On the one hand, the expression regulation of GSDMs lacks tumor specificity, potentially leading to severe off-target effects. On the other hand, the precise spatiotemporal control of pyroptosis induction still needs optimization to ensure that damage to normal tissues is minimized while maximizing antitumor efficacy. Therefore, developing precise pyroptosis induction strategies based on tumor-specific promoters or microenvironment-responsive delivery systems is of great significance for improving the safety of pyroptosis immunotherapy.

[0004] Tumor-specific promoters (TSPs) are a class of transcriptional regulatory elements that exhibit high transcriptional activity in tumor cells but remain quiescent in normal tissues. Their mechanism of action primarily relies on the aberrant activation of tumor-specific epigenetic modifications (such as DNA hypomethylation) or transcription factors (such as c-Myc and NF-κB), enabling spatiotemporally specific expression of therapeutic genes and significantly reducing off-target toxicity to healthy tissues. For example, the human telomerase reverse transcriptase (hTERT) promoter, a classic tumor-targeting regulatory element, has become an important tool in tumor gene therapy due to its aberrant activation in over 90% of malignant tumors (positively correlated with telomerase activity). In adenovirus-mediated gene therapy research, the hTERT promoter has been shown to have strong activating effects in various human cancer cell lines, while its activity is extremely low in normal human cells. Notably, although the telomerase regulatory mechanism in rodents differs significantly from that in humans, the tumor-specific transcriptional activity of the hTERT core promoter region is also applicable in mouse cells, providing an important experimental basis for translational evaluation in preclinical studies. However, tumor-specific promoters generally suffer from low transcriptional activity, making it necessary to enhance transcriptional activity by optimizing promoter sequences or employing transcription amplification strategies to improve the effectiveness of gene therapy.

[0005] Besides the transcriptional activity limitations imposed by tumor-specific promoters, cells possess various molecular mechanisms that inhibit pyroptosis, which may also negatively impact the effectiveness of pyroptosis gene therapy. For example, in a 2018 research paper published in Science, Sebastian Rühl et al. found that the membrane remodeling ESCRT-III (Endosomal Sorting Complexes Required for Transport III) complex can be recruited to the plasma membrane for membrane repair after GSDMD activation, thereby limiting the secretion of pro-inflammatory cytokines and the occurrence of pyroptosis. Furthermore, in a 2020 Science research paper, Fiachra Humphries et al. found that fumarate produced during metabolism can modify GSDMD and GSDME at specific cysteine ​​sites, thereby inhibiting their oligomer formation ability and preventing pyroptosis.

[0006] In summary, developing a pyroptosis-inducing system based on tumor-specific promoter transcriptional activity that can specifically and effectively function in tumor cells has significant application prospects in improving the safety and applicability of pyroptosis immunotherapy. Summary of the Invention

[0007] The purpose of this invention is to provide a tumor-specific pyroptosis induction system based on the principle of transcriptional amplification and its application in the preparation of antitumor drugs.

[0008] The technical solution adopted in this invention is:

[0009] A tumor-specific pyroptosis induction system based on transcriptional amplification includes a first expression vector and a second expression vector:

[0010] (a) The first expression vector contains a tumor-specific promoter and a site-specific recombinase gene regulated by the tumor-specific promoter;

[0011] (b) The second expression vector comprises a strong promoter, a pyroptosis-inducing protein gene, and a transcriptional blocking element disposed between the strong promoter and the pyroptosis-inducing protein gene; the transcriptional blocking element consists of two co-located recombination sites that can be specifically recognized by the site-specific recombinase, and a transcription termination sequence disposed between the two recombination sites.

[0012] Furthermore, the first expression vector and the second expression vector work synergistically: in tumor cells, the tumor-specific promoter is transcribed and activated, driving the expression of the site-specific recombinase gene; the site-specific recombinase recognizes the recombination site and removes the transcription termination sequence between the two recombination sites, thereby relieving transcriptional blockade, and the strong promoter drives the efficient expression of the pyroptosis-inducing protein gene, thereby specifically inducing pyroptosis in tumor cells.

[0013] Furthermore, the aforementioned tumor-specific promoters are selected from one or more of the following: human telomerase reverse transcriptase promoter, survivin promoter, alpha-fetoprotein promoter, carcinoembryonic antigen promoter, and prostate-specific antigen promoter.

[0014] Furthermore, the site-specific recombination system formed by the above-mentioned site-specific recombinase and the corresponding recombination site is selected from one or more of the Cre-LoxP system, FLP-FRT system, Dre-Rox system, VCre-VloxP system, SCre-SloxP system, and ΦC31 integrase system.

[0015] Furthermore, the aforementioned strong promoters are selected from one or more of the following: MCMV promoter, CMV promoter, EF1α promoter, CAG promoter, PGK promoter, UBC promoter, and SV40 promoter.

[0016] Furthermore, the aforementioned pyroptosis-inducing proteins are selected from one or more of the pore-forming active fragments of Gasdermin family proteins and upstream activators of the pyroptosis pathway; Gasdermin family proteins include GSDMA, GSDMB, GSDMC, GSDMD, and GSDME.

[0017] Furthermore, the first expression vector and the second expression vector are jointly delivered to the target cells via gene delivery methods. The gene delivery method is selected from one or more of electroporation, chemical delivery, and viral vector delivery. Chemical delivery methods include cationic liposomes, lipid nanoparticles, calcium phosphate precipitation, DEAE-dextran, polygluconate, and nanomaterial delivery. Viral vectors include virus-like particles, adenovirus, adeno-associated virus, lentivirus, and retrovirus.

[0018] The above-mentioned tumor-specific pyroptosis induction system is used in the preparation of antitumor drugs.

[0019] Furthermore, the administration method of the above-mentioned antitumor drugs is selected from one or more of intratumoral injection, intravenous injection, and local administration to the surgical resection wound of the tumor; the antitumor drugs are used to inhibit the growth of solid tumors, inhibit distant metastasis of tumors, and / or inhibit postoperative recurrence of tumors.

[0020] An antitumor drug comprising the aforementioned tumor-specific pyroptosis induction system and a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier comprising a hydrogel, which may be a natural hydrogel or a synthetic hydrogel; the natural hydrogel includes collagen, gelatin, hyaluronic acid, chitosan, fibrin, and alginate hydrogel, and the synthetic hydrogel includes hydrogels formed by crosslinking polyacrylic acid and its derivatives, polyvinyl alcohol, polyoxyethylene, and polyacrylamide.

[0021] The beneficial effects of this invention are mainly reflected in:

[0022] (1) The use of tumor-specific promoters in this invention can limit the expression of pyroptosis-inducing proteins to tumor cells, reduce the risk of normal cells being damaged by pyroptosis therapy, and improve the safety of tumor pyroptosis therapy.

[0023] (2) The transcription amplification system constructed based on site-specific recombinase and recombinase recognition site in this invention drives recombinase expression through tumor-specific promoter. After the transcription termination sequence is removed, the expression of pyroptosis-induced protein is driven by a strong promoter. This overcomes the limitation of the weak transcriptional activity of the tumor-specific promoter itself, ensures the efficient expression of pyroptosis-induced protein in tumor cells, and guarantees the effectiveness of tumor pyroptosis therapy.

[0024] (3) The enhanced safety of treatment in this invention helps to increase the upper limit of gene therapy dosage and thus enhance the effectiveness of pyroptosis treatment. It can also effectively expand the application scope of pyroptosis in tumor treatment (for example, after tumor surgical resection, it can be used to treat wounds and prevent tumor recurrence).

[0025] In summary, the system described in this invention is beneficial for improving the safety and effectiveness of pyroptosis therapy for tumors, and on this basis, expands the scope of application of pyroptosis therapy. Attached Figure Description

[0026] Figure 1 This study aimed to construct and validate a tumor-specific pyroptosis induction system. A shows a schematic diagram of the system's action; B shows the vector structure and abbreviations; C shows the expression level of the luciferase reporter gene; and D shows the Western blot analysis of FLP and GSDMD after transfection of HEK293T cells with the vector. NT Level of expression.

[0027] Figure 2 This study describes the preparation and functional validation of adenovirus delivery vectors. A shows schematic diagrams of control and pyroptosis-inducing adenovirus vectors; B shows schematic diagrams of adenovirus combinations; and C shows the pyroptosis-inducing effects of different titers of adenovirus combinations in normal cell lines (HUVEC and BALB / 3T3 cells) and tumor cell lines (Hepa1-6, 4T1, SK-Hep-1, and SMMC-7721 cells).

[0028] Figure 3 The therapeutic effect of the pyroptosis-inducing vector system delivered intratumorally via an adenovirus vector is shown. In the figure, A represents the timeline of intratumoral treatment in mice; B represents the monitoring results of tumor growth by in vivo imaging in mice; C represents the tumor growth curve on the injection side; and D represents the tumor growth curve at the distal end.

[0029] Figure 4 The therapeutic effect of the pyroptosis-inducing vector system delivered via adenovirus vector in a tumor resection model is shown. A represents the timeline of tumor resection model treatment; B represents the monitoring results of tumor recurrence and metastasis in mice using in vivo imaging; C represents the size of the in situ recurrent tumor after mouse sacrifice; and D represents the monitoring results of luciferase activity in distal lung metastases. Detailed Implementation

[0030] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0031] Example 1: Construction and validation of a tumor-specific pyroptosis induction system based on transcription amplification principle

[0032] This embodiment constructs a transcription amplification system based on the adenovirus vector pDC315 (SEQ ID NO.1) and verifies in vitro that this system can specifically enhance the pyroptosis core protein GSDMD in tumor cells. NT The level of expression.

[0033] In this invention, GOI is a general abbreviation for Gene of Interest, which can be replaced with a functional gene or reporter gene according to experimental needs. This embodiment and... Figure 1 The terms TERT-GOI, MCMV-GOI, and MF-GOI mentioned are all unified names for the corresponding target gene vectors.

[0034] The system works in tandem with two functional carriers:

[0035] Construction method of pDC315-TF (TF vector for short): The original MCMV promoter of pDC315 backbone is replaced with hTERT promoter (SEQ ID NO.2), while retaining the original SV40 polyA of pDC315 backbone; the FLP recombinase gene (SEQ ID NO.3) is inserted into the multiple cloning site between hTERT promoter and SV40 polyA to form a complete expression cassette.

[0036] The pDC315-MF-GSDMD vector (abbreviated as MF-GSDMD vector, also known as MF-GOI vector) is constructed as follows: The original MCMV promoter and SV40 polyA of the pDC315 backbone are retained; two cis-FRT sites and their sandwiched polyA transcription termination sequence (i.e., FRT-polyA-FRT element, SEQ ID NO.4), and GSDMD are added. NT The gene (SEQ ID NO.5) is sequentially inserted into the multiple cloning site between the MCMV promoter and SV40 polyA to form a complete expression cassette. Among them, GSDMD... NT The gene is located downstream of the entire FRT-polyA-FRT element.

[0037] The mechanism of action is as follows: Transcriptional activation of the hTERT promoter in tumor cells specifically drives the expression of the FLP recombinase; FLP recognizes the FRT site and cleaves the intermediate polyA transcription termination sequence, thus releasing the transcriptional blockade, allowing the MCMV promoter to continuously and efficiently drive GSDMD. NT The pDC315 backbone contains SV40 polyA, which ensures stable mRNA translation and ultimately induces pyroptosis in tumor cells.

[0038] The experiment included two control vectors:

[0039] The negative control vector TERT-GSDMD (also known as TERT-GOI) is constructed in the same manner as pDC315-TF, but the FLP recombinase gene is replaced with GSDMD. NT The gene, GSDMD, is driven by the hTERT promoter. NT Express;

[0040] The positive control vector MCMV-GSDMD (also known as MCMV-GOI) is constructed in the same way as pDC315-MF-GSDMD, but the FRT-polyA-FRT element is omitted, and GSDMD is directly driven by the MCMV promoter. NT Express.

[0041] To verify the transcriptional activation efficiency of the transcription amplification system, the TERT-GSDMD, MCMV-GSDMD, and pDC315-MF-GSDMD vectors were modified, respectively, to convert GSDMD... NTThe gene was replaced with the luciferase reporter gene Luc, resulting in three reporter vectors. Specifically, the TERT-GSDMD vector was modified to obtain the TERT-Luc vector, the MCMV-GSDMD vector was modified to obtain the MCMV-Luc vector, and the pDC315-MF-GSDMD vector was modified to obtain the MF-Luc vector. Each vector was transfected into HEK293T cells, with the TF+MF-Luc group treated by co-transfection with the TF and MF-Luc vectors. After 24 hours of continuous culture, cells were collected, and luciferase activity was measured using a luciferase assay kit (TransGen Biotech, FR101-02-V2) according to the manufacturer's instructions to analyze reporter gene expression levels. Results showed ( Figure 1 (C) Compared to the TERT-Luc group, the reporter gene expression level in the MCMV-Luc group was about 100-fold higher; the reporter gene expression level in the TF+MF-Luc group was comparable to that in the MCMV-Luc group.

[0042] GSDMD was further detected using Western blot. NT Protein expression levels. HEK293T cells were transfected with TF vector, MF-GSDMD vector, TERT-GSDMD vector, TF+MF-GSDMD co-transfection group, and MCMV-GSDMD vector, respectively. Cells were collected 24 hours after transfection, and total protein was extracted. Protein samples were separated by SDS-PAGE and transferred to NC membranes for detection of Flag-tagged GSDMD. NT HA-tagged FLP recombinase was detected using anti-HA antibody, and anti-β-actin antibody was used as an internal control for immunoblotting detection. Results showed ( Figure 1 D) After transfection with HEK293T cells, GSDMD was undetectable in the TF group and the MF-GSDMD group. NT The expression of GSDMD in the TERT-GSDMD group. NT The expression level was low; while the expression levels of GSDMD in the TF+MF-GSDMD co-transfection group and the MCMV-GSDMD group were lower. NT The expression levels were comparable, but significantly higher than those in the TERT-GSDMD group.

[0043] The above results confirm that the gene transcription efficiency mediated by the hTERT promoter alone is low, while the transcription amplification system constructed based on FLP / FRT recombinase can significantly improve the efficiency of GSDMD regulated by hTERT. NT The protein expression level provides the molecular basis for inducing potent pyroptosis in tumor cells.

[0044] Example 2: Preparation of recombinant adenovirus carrying a transcription amplification system and in vitro verification of its cell-specific killing function.

[0045] Adenovirus type 5 (Ad5) has high gene transduction efficiency and can exert an in situ immune adjuvant effect, making it suitable for in vivo gene delivery. In this embodiment, the AdMax adenovirus packaging system was used to package recombinant adenovirus based on the recombinant shuttle vector constructed in Example 1, and its pyroptosis-inducing specificity and killing effect were verified in normal cells and tumor cells.

[0046] The adenovirus backbone plasmid was pBHGlox (delta) E1,3 Cre. The recombinant shuttle vectors pDC315-TF and pDC315-MF-GSDMD constructed in Example 1 were co-transfected with the adenovirus backbone plasmid into HEK293A cells. Recombinant adenoviruses Adv-TF ​​and Adv-MF-GSDMD were packaged using the Cre / loxP site-specific homologous recombination mechanism of the AdMax system. Simultaneously, the empty shuttle vector pDC315 was co-transfected with the adenovirus backbone plasmid to obtain the blank control adenovirus Adv-Ctrl. All recombinant adenoviruses were amplified, purified, and titered before being used in subsequent experiments. Figure 2 A).

[0047] like Figure 2 As shown in B, the experiment set up four virus treatment combinations: Adv-Ctrl+Adv-Ctrl group, Adv-Ctrl+Adv-MF-GSDMD group, Adv-Ctrl+Adv-TF ​​group, and Adv-TF+Adv-MF-GSDMD group. In each group, the two types of viruses were mixed at a virus particle ratio of 1:1. Normal cells (human umbilical vein endothelial cells HUVEC, mouse embryonic fibroblasts BALB / 3T3clone A31) and tumor cells (mouse hepatocellular carcinoma cells Hepa1-6, mouse breast cancer cells 4T1, human hepatic sinusoidal endothelial carcinoma cells SK-Hep-1, and human hepatocellular carcinoma cells SMMC-7721) were infected with different multiplicity of infection (MOI) (0, 10, 20, 30, 40, 50, where MOI is the multiplicity of infection of a single virus in the mixed virus). Twenty-four hours after viral infection, cell culture supernatant and cell lysate were collected separately. LDH activity was detected using a lactate dehydrogenase (LDH) cytotoxicity assay kit (Beyotime, C0017), and LDH release rate was calculated to assess the degree of cell membrane integrity disruption and pyroptosis-related cytotoxicity.

[0048] The results show that ( Figure 2 C) In normal cells, none of the four virus combinations caused significant LDH release or cell damage under any MOI condition; in tumor cells, compared with the control groups, the Adv-TF+Adv-MF-GSDMD group was able to significantly increase LDH release in a dose-dependent manner.

[0049] These results demonstrate that this adenovirus-delivered tumor-specific pyroptosis induction system can efficiently and specifically induce pyroptosis in tumor cells.

[0050] Example 3: Therapeutic effect of tumor-specific pyroptosis induction system delivered intratumorally via adenovirus vector

[0051] In this embodiment, a 4T1-Luc bilateral subcutaneous tumor-bearing mouse model was constructed to evaluate the in vivo antitumor effect of an adenovirus-loaded tumor-specific pyroptosis-inducing system.

[0052] Female BALB / c mice aged 6-8 weeks and weighing 20-25g were selected. 4T1-Luc stably transfected breast cancer cells were resuspended in an equal volume mixture of 1×PBS (pH 7.4) and Matrigel (Corning Life Sciences, catalog number 3330624). The total cell inoculation amount per mouse was 2×10⁶ cells. 6 100 μl total volume of cells were injected subcutaneously into the left and right sides of the back of mice (i.e., 1 × 10⁶ cells were injected per side). 6 (Each injection, 50 μl).

[0053] Intratumoral injection therapy was initiated on day 7 (d7) after cell seeding. A 2% silk fibroin precursor solution was prepared according to the method described in reference PMID: 21959241. The solution was pretreated with ultrasound, with the ultrasound parameters set to 30% amplitude, pulse mode (2 seconds on, 3 seconds off), and a total ultrasound duration of 3 minutes. This ultrasound treatment induced a conformational change in the silk fibroin, initiating a cross-linking process and gradual gelation, exhibiting a gelation lag time window. Utilizing this window, Adv-TF ​​and Adv-MF-GSDMD were mixed at a 1:1 ratio, with a total viral dose of 2 × 10⁻⁶ for a single injection. 8 PFU was mixed thoroughly with the ultrasound-treated silk fibroin precursor solution; 50 μl was injected intratumorally into the right tumor of each mouse. Administration was performed on days 7, 9, 11, and 13, for a total of four intratumoral injections.

[0054] From day 7 to day 29, tumor volume was measured every other day using calipers; a parallel batch of mice was also set up and treated with the same administration regimen, and the survival of the mice was continuously observed until day 60 after inoculation.

[0055] Tumor growth monitoring results showed ( Figure 3 In A-3D mice, an intratumoral injection of an Adv-TF+Adv-MF-GSDMD virus combination encapsulated in silk protein hydrogel not only effectively inhibited the growth of the primary tumor on the injection side, but also significantly reduced the volume of the distal tumor on the contralateral side. Some mice even achieved complete tumor remission, demonstrating that this pyroptosis-inducing system has excellent in vivo tumor treatment effects.

[0056] Example 4: Therapeutic effect of tumor-specific pyroptosis induction system delivered via adenovirus vector in a tumor resection model.

[0057] This embodiment constructs a surgical resection model of breast cancer in situ tumors to simulate a clinical adjuvant therapy scenario after tumor surgery and explore the system's ability to inhibit tumor recurrence in situ and distant metastasis.

[0058] like Figure 4 As shown in Figure A, female BALB / c mice aged 6-8 weeks and weighing 20-25g were selected. 4T1-Luc stable transgenic breast cancer cells were resuspended in an equal volume mixture of 1×PBS (pH 7.4) and Matrigel. The total cell inoculation amount per mouse was 2×10⁻⁶ cells. 6 Cells (total volume 100 μl) were in situ injected into the abdominal fat pads of mice to construct in situ mammary tumors. The first in vivo fluorescence imaging was performed on day 14 (d14) post-inoculation, and surgery was performed on day 15 (d15) to remove approximately 2 / 3 of the in situ tumor tissue. Immediately after surgical wound closure, the drug was injected into the tissue cavity formed by tumor resection. The drug administration groups were set as follows: The treatment group received ultrasonic pretreatment with the same 2% silk fibroin precursor solution formulation as in Example 3, with ultrasonic parameters of 30% amplitude, pulse mode (2 seconds on, 3 seconds off), and a total ultrasonic duration of 3 minutes. This ultrasonic treatment induced a conformational change in silk fibroin, and the system subsequently initiated a cross-linking process and gradually gelled. A gelation lag time window existed, during which Adv-TF ​​and Adv-MF-GSDMD were mixed at a 1:1 ratio and added to the precursor solution. The total viral dose per dose was 2 × 10⁻⁶. 9 PFU was injected in a total volume of 50 μl. The control group received the same volume of silk fibroin gel preparation as the treatment group, but with 1×PBS (pH 7.4) instead of the virus mixture. In vivo fluorescence imaging was performed again on day 21 (d21) and day 42 (d42) after inoculation to dynamically monitor the occurrence of in situ tumor recurrence and distant metastasis in mice.

[0059] Statistical results of live imaging show that ( Figure 4 Mice treated with Adv-TF+Adv-MF-GSDMD gel in the surgical cavity (B-4D) showed significantly lower rates of in situ tumor recurrence and distant lung metastasis compared to the control group, demonstrating the therapeutic effectiveness of this tumor-specific pyroptosis induction system in preventing postoperative recurrence and metastasis.

[0060] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A tumor-specific pyroptosis induction system based on transcriptional amplification, characterized in that: Including the first expression vector and the second expression vector: (a) The first expression vector contains a tumor-specific promoter and a site-specific recombinase gene regulated by the tumor-specific promoter; (b) The second expression vector comprises a strong promoter, a pyroptosis-inducing protein gene, and a transcriptional blocking element disposed between the strong promoter and the pyroptosis-inducing protein gene; the transcriptional blocking element consists of two co-located recombination sites that can be specifically recognized by the site-specific recombinase, and a transcription termination sequence disposed between the two recombination sites.

2. The tumor-specific pyroptosis induction system according to claim 1, characterized in that: The first expression vector and the second expression vector work synergistically: In tumor cells, the tumor-specific promoter is transcribed and activated, driving the expression of the site-specific recombinase gene; the site-specific recombinase recognizes the recombination site and removes the transcription termination sequence between the two recombination sites, thereby relieving the transcriptional blockade, and the strong promoter drives the efficient expression of the pyroptosis-inducing protein gene, thereby specifically inducing pyroptosis in tumor cells.

3. The tumor-specific pyroptosis induction system according to claim 1, characterized in that: The tumor-specific promoter is selected from one or more of the following: human telomerase reverse transcriptase promoter, survivin promoter, alpha-fetoprotein promoter, carcinoembryonic antigen promoter, and prostate-specific antigen promoter.

4. The tumor-specific pyroptosis induction system according to claim 1, characterized in that: The site-specific recombinase and the corresponding recombination site constitute a site-specific recombination system selected from one or more of the Cre-LoxP system, FLP-FRT system, Dre-Rox system, VCre-VloxP system, SCre-SloxP system, and ΦC31 integrase system.

5. The tumor-specific pyroptosis induction system according to claim 1, characterized in that: The strong promoter is selected from one or more of the following: MCMV promoter, CMV promoter, EF1α promoter, CAG promoter, PGK promoter, UBC promoter, and SV40 promoter.

6. The tumor-specific pyroptosis induction system according to claim 1, characterized in that: The pyroptosis-inducing protein is selected from one or more of the pore-forming active fragments of Gasdermin family proteins and upstream activators of the pyroptosis pathway; the Gasdermin family proteins include GSDMA, GSDMB, GSDMC, GSDMD, and GSDME.

7. The tumor-specific pyroptosis induction system according to claim 1, characterized in that: The first expression vector and the second expression vector are jointly delivered to the target cells via gene delivery methods; the gene delivery method is selected from one or more of electroporation, chemical delivery, and viral vector delivery; the chemical delivery method includes delivery via cationic liposomes, lipid nanoparticles, calcium phosphate precipitation, DEAE-dextran, polygluconate, and nanomaterials; the viral vector includes virus-like particles, adenovirus, adeno-associated virus, lentivirus, and retrovirus.

8. The use of the tumor-specific pyroptosis induction system according to any one of claims 1 to 7 in the preparation of antitumor drugs.

9. The application according to claim 7, characterized in that: The administration method of the antitumor drug is selected from one or more of intratumoral injection, intravenous injection, and local administration to the surgical resection wound of the tumor; the antitumor drug is used to inhibit the growth of solid tumors, inhibit distant metastasis of tumors, and / or inhibit postoperative recurrence of tumors.

10. An antitumor drug, characterized in that: The invention comprises the tumor-specific pyroptosis induction system according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier comprises a hydrogel, which is a natural hydrogel or a synthetic hydrogel; the natural hydrogel includes collagen, gelatin, hyaluronic acid, chitosan, fibrin, and alginate hydrogel, and the synthetic hydrogel includes hydrogels formed by crosslinking polyacrylic acid and its derivatives, polyvinyl alcohol, polyoxyethylene, and polyacrylamide.

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