A method for constructing an animal model of glioma based on AAV-mediated immune microenvironment

Abstract

The present invention discloses a method for constructing an animal model of glioma based on AAV-mediated immune microenvironment, comprising: selecting immunodeficient mice of appropriate age; injecting human CD34+ hematopoietic stem cells for immune system reconstruction; detecting human CD45+ immune cells in peripheral blood PBMC; selecting tumor tissues that highly express glioma marker genes, performing rapid qPCR or targeted RNA-seq immunohistochemistry multiple verification, and screening tumor tissues that meet the requirements; physically cutting the tumor tissue and treating it with a pretreatment solution, digesting it into a single cell suspension, and inoculating it into the mouse's skull; injecting an AAV vector carrying a specific immune regulatory gene into the mouse's tail vein to regulate the glioma immune microenvironment; when the tumor tissue is observed to grow stably and have immune cell infiltration characteristics, the construction of the glioma animal model is completed. Compared with existing models, the present invention provides a more stable, more scientific, and more human immune background glioma model, which is suitable for related immunotherapy drug screening and mechanism research.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor animal models, and in particular to a method for constructing a brain glioma animal model based on AAV-mediated immune microenvironment. Background Art

[0002] Glioblastoma (GBM) is a highly invasive and malignant brain tumor that typically originates from astrocytes in the brain or spinal cord. GBM is the most common primary brain tumor in adults and one of the most lethal. The disease is characterized by rapid proliferation, high infiltrative capacity, resistance to conventional therapies (such as radiotherapy and chemotherapy), and a high recurrence rate after treatment. The pathogenesis of GBM is complex, involving multiple molecular pathways and gene mutations, and the tumor microenvironment (such as immune escape mechanisms and blood-brain barrier disorders) has a significant impact on tumor progression. Therefore, it is crucial to study the mechanisms of occurrence and development of gliomas and to optimize existing treatment strategies.

[0003] Glioma research relies on a variety of experimental models, including in vitro cell culture, subcutaneous xenograft models, genetically engineered mouse models (GEMMs), and orthotopic glioma models. While these models have played an important role in research, they also have technical limitations. In vitro cell culture models fail to mimic the tumor microenvironment (TME) and lack the interactions between immune cells and blood vessels, resulting in an inability to faithfully reproduce tumor immune escape mechanisms and growth characteristics. Subcutaneous xenograft models, while useful for drug screening, fail to reflect glioma growth and drug response due to microenvironmental differences from brain tissue. Genetically engineered mouse models mimic tumor development through genetic modification, but their long development time, limited specificity of genetic modification, and inability to fully recapitulate tumor heterogeneity and microenvironmental limitations restrict their application in drug screening. Orthotopic glioma models can better simulate the brain microenvironment, but they still face challenges such as inaccurate tumor cell injection, incomplete preservation of the tumor microenvironment, inadequate simulation of the immune microenvironment, and a lack of spontaneous metastasis. Therefore, the limitations of these existing models pose challenges to glioma drug development and clinical translation, particularly with regard to the accuracy of immunotherapy and drug screening.

[0004] Therefore, it is particularly important to develop an animal model that can overcome the limitations of existing glioma experimental models. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for constructing an animal model of glioma based on AAV-mediated immune microenvironment to solve one or more technical problems existing in the prior art and at least provide a beneficial option or create conditions.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned purpose:

[0007] The present invention provides a method for constructing an animal model of glioma based on AAV-mediated immune microenvironment, comprising:

[0008] Select immunodeficient mice of appropriate age;

[0009] Immune system reconstitution via tail vein injection of human CD34+ hematopoietic stem cells;

[0010] Flow cytometry was used to detect human CD45+ immune cells in peripheral blood PBMCs to confirm the successful reconstitution of the immune system;

[0011] Select tumor tissues that highly express glioma marker genes and perform rapid qPCR or targeted RNA-seq immunohistochemistry multiplex validation to screen tumor tissues that are consistent with glioma animal models;

[0012] The selected tumor tissues were physically cut into pieces and treated with a pretreatment solution before being digested into single-cell suspensions, which were then inoculated into the mouse skull using stereotactic injection.

[0013] Injecting an AAV vector carrying a specific immune regulatory gene into the tail vein of mice to regulate the immune microenvironment of brain gliomas. When stable expression of the AAV expression product is detected in the mice, the immune regulation mediated by the AAV expression product is completed.

[0014] When the tumor tissue is observed to grow stably and have immune cell infiltration characteristics, it is confirmed that the immune microenvironment reconstruction and tumor growth meet the requirements of model construction, and the construction of the brain glioma animal model is completed.

[0015] Furthermore, the AAV vector includes an AAV-TGFβ1-P2A-IL10 expression cassette and an AAV-CSF1 expression cassette for expressing TGF-β1 、 IL10 and CSF1 Gene;

[0016] The AAV-TGFβ1-P2A-IL10 expression cassette includes a CAG promoter driven by TGF-β1 Genes and IL10 The nucleic acid structure of the gene co-expressed by the P2A self-cleavage peptide sequence has a total nucleic acid sequence length of 3.1 ± 0.2 kb;

[0017] The AAV-CSF1 expression cassette includes a CAG promoter driven by CSF1 Gene expression unit, the total length of nucleic acid sequence is 2.3±0.2 kb.

[0018] Furthermore, AAV9 or AAV-PHP.eB serotype was used for TGF-β1 、 IL10 and CSF1 Gene delivery.

[0019] Furthermore, the injection dose of the AAV vector is 1-5E+11 vg / mouse;

[0020] The AAV vector is formulated in a buffer solution with a pH of 7.7 to 8.3, comprising 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20.

[0021] Furthermore, the tumor tissue is selected from tumor tissue that is pathologically confirmed to be WHO grade III or IV.

[0022] Furthermore, the brain glioma marker genes include CD133 protein, IDH1 R132H mutation, EGFR amplification, MGMT promoter methylation and SOX2 protein.

[0023] Furthermore, the pretreatment solution includes Neurobasal culture medium and Matrigel mixed in a volume ratio of 3:1, and is supplemented with 2× B27, 2 mM glutathione, 50 U / mL penicillin-streptomycin, and 20 ng / mL PDGF-BB.

[0024] Furthermore, the growth monitoring of the tumor tissue is obtained by combining T2-weighted images of small animal MRI with volume change calculation.

[0025] Furthermore, the morphology of the tumor cells is observed by HE staining.

[0026] Furthermore, the method for detecting significant expression of AAV expression products in mice includes:

[0027] qPCR technology was used to detect the expression level of mRNA;

[0028] Western Blot or ELISA techniques were used to detect protein expression levels;

[0029] Immunohistochemical staining or immunofluorescence staining was used to further verify the transgene expression product in the tissue.

[0030] The beneficial effects of the present invention are as follows:

[0031] The present invention should not only be able to more accurately simulate the tumor microenvironment, immune escape mechanism and tumor growth characteristics of brain glioma, but also have the advantages of good repeatability and rapid construction to improve the success rate of drug screening and clinical transformation.

[0032] This invention fully considers the defects, deficiencies and usage limitations of existing glioma models. It uses humanized mice and patient-derived glioma tissue and its appendages as research objects. It uses AAV vectors to long-term express inflammatory factors related to the immune microenvironment of glioma, regulates the immune microenvironment of the mouse glioma model, optimizes immune checkpoint expression, and establishes a mouse glioma model that is closer to the immune characteristics of human glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 An overall flow chart provided according to an embodiment of the present invention;

[0034] Figure 2 This is a flow cytometry measurement result of mouse immune system reconstruction by tail vein injection of human CD34+ hematopoietic stem cells according to an embodiment of the present invention;

[0035] Figure 3 This is a graph showing the growth curve of the tumor in the mouse brain measured by MRI after transplantation of a single cell suspension obtained by digestion of brain glioma tissue according to an embodiment of the present invention;

[0036] Figure 4 This is a diagram showing the expression of vector mRNA in peripheral blood detected by qPCR technology after AAV injection according to an embodiment of the present invention;

[0037] Figure 5 This is a diagram showing the expression of vector mRNA in tumor tissues detected by qPCR technology after AAV injection according to an embodiment of the present invention;

[0038] Figure 6 This is a graph showing the protein expression of human TGF-β1, IL-10, and CSF1 in tumor tissue after AAV immune system reconstruction verified by the IHC method provided in an embodiment of the present invention;

[0039] Figure 7 A diagram showing the dynamic changes of brain glioma in mice under MRI monitoring according to different glioma construction factors provided by an embodiment of the present invention;

[0040] Figure 8 A diagram of a flow cytometry antibody combination according to an embodiment of the present invention;

[0041] Figure 9 This is a diagram showing the quantitative verification results of immune reconstitution of human CD34+ mesenchymal stem cells according to an embodiment of the present invention;

[0042] Figure 10 This is a graph showing the effects of different immune reconstitution conditions on the intracranial growth curve of mice after brain glioma tissue cell transplantation according to an embodiment of the present invention;

[0043] Figure 11A diagram showing the selection and function of antibodies provided according to an embodiment of the present invention;

[0044] Figure 12 This is a graph showing the changes in the immune microenvironment of tumor tissues under different reconstruction conditions measured by flow cytometry according to an embodiment of the present invention;

[0045] Figure 13 Schematic diagram of the design structure of the AAV-TGFβ1-P2A-IL10 vector provided according to an embodiment of the present invention;

[0046] Figure 14 Schematic diagram of the AAV-CSF1 vector design structure provided according to an embodiment of the present invention. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0048] like Figures 1-14 As shown, the present invention provides a method for constructing an animal model of glioma based on AAV-mediated immune microenvironment, and the specific steps are as follows:

[0049] S1. Mouse selection and housing conditions

[0050] This study used NOG (NOD.Cg- Prkdc scid Il2rg tm1Sug JicCrl mice were used as experimental animals. This strain of mice is severely immunodeficient and suitable for immune system reconstitution. Three-week-old female mice were used in this experiment. The mice were housed in an SPF-rated barrier environment with a controlled temperature of 22-26°C, a humidity of 40-60%, and a 12-hour light / 12-hour dark photoperiod. The mice were fed a sterilized, specific pathogen-free (SPF) diet and ultrafiltered sterilized water. These conditions ensured that the mice maintained a suitable environment for growth and experimentation.

[0051] S2, human immune system reconstitution

[0052] In the experiment, human CD34+ hematopoietic stem cells were used to reconstitute the immune system. CD34+ cells can be isolated from human umbilical cord blood or bone marrow and sorted by FACS to obtain CD34+ cells with a purity greater than 95%. The cell concentration was set at 1-5×10^6 cells / 100 μL PBS. A 30G fine needle was used for tail vein injection, and the injection should be performed slowly to ensure smooth cell infusion. Monitoring of immune system reconstitution is usually performed after 4-6 weeks, and immune cell markers in peripheral blood, such as human CD45+, CD3+, and CD19+, are detected by flow cytometry. If the proportion of human CD45+ cells in peripheral blood cells exceeds 25%, the immune system is considered to have been reconstituted successfully.

[0053] Here, the immune system of mice was successfully reconstructed by tail vein injection of human CD34+ hematopoietic stem cells, which can more realistically simulate the human immune microenvironment, especially in the study of immune escape and immunotherapy of glioma, providing an immune response closer to that of humans. At the same time, most existing glioma mouse models are immunodeficient mouse models and cannot simulate normal immune responses. However, by establishing an immune reconstitution model with a complete immune system, this application enables the model to better evaluate immunotherapy and immune escape mechanisms, increasing the relevance and translational potential of clinical research.

[0054] S3, Tumor marker gene screening and validation

[0055] Before establishing a tumor model, select tumor tissue that highly expresses brain glioma marker genes. Human glioblastoma (GBM, WHO grade III or IV) tissue was selected for qPCR or RNA-seq detection. The marker genes screened include CD133 、 IDH1 、 EGFR 、 MGMT 、 SOX2 、 SLC1A3 、 OLIG2 Its TPM value must meet the following requirements (any three of the following indicators meet the standards):

[0056] CD133>80

[0057] IDH1>50

[0058] EGFR>50

[0059] MGMT>30

[0060] SOX2>100

[0061] SLC1A3>100

[0062] OLIG2>25

[0063] In addition, the protein expression level was further verified by immunohistochemistry (IHC) to ensure that the selected tumor tissues had high gene expression levels.

[0064] S4, tumor tissue pretreatment

[0065] After screening for tumor tissue that meets the criteria, the tissue was cut into 1-3 mm³ pieces. To ensure the biological activity of the tissue pieces, a specific pretreatment method was used. The pretreatment solution consisted of a mixture of Neurobasal medium and Matrigel at a volume ratio of 3:1, supplemented with 2× B27, 2 mM glutathione, 50 U / mL penicillin-streptomycin, and 20 ng / mL PDGF-BB. The tissue was then centrifuged at low speed to remove necrotic tissue, and digested with a cocktail of enzymes. After digestion, the tissue was washed twice with PBS to terminate the digestion process and prepare for transplantation.

[0066] Here, an optimized tumor tissue pretreatment method, including low-speed centrifugation to remove necrotic tissue, moderate digestion, and PBS washing, is used to ensure the viability and cell survival of transplanted tumor tissue. Traditional tumor transplantation methods may suffer from tissue necrosis or low cell survival. This carefully optimized tissue pretreatment method can ensure tumor cell viability, improve the growth rate and success rate of transplanted tumors, and enhance the reproducibility and reliability of the model.

[0067] S5, tissue transplantation

[0068] After pretreatment, tumor tissue explants were transplanted into the brains of immunodeficient mice. During the transplantation procedure, mice were anesthetized with isoflurane inhalation at an induction concentration of 3% and a maintenance concentration of 1.5%. Under aseptic technique, the mouse head was immobilized, and the scalp was incised to expose the skull. Using a stereotaxic injection technique, a dental microdrill was used to drill a hole in the left or right frontal lobe (2 mm posterior to bregma and 2 mm left or right of the midline) at a depth of 2.5–3 mm. Following transplantation, the skull hole was sealed, the scalp was sutured, and antibiotics (cefotaxime 25 mg / kg, ip) were administered for infection prevention.

[0069] S6, AAV vector injection and immune microenvironment regulation

[0070] To modulate the immune microenvironment of gliomas, AAV vectors containing specific genes are injected into the tail vein of model animals. The AAV packaging capacity is limited to 4.7 kb, so the length of the gene element in the AAV vector must be strictly calculated. Specific AAV vector designs are: AAV-TGFβ1-P2A-IL10 (approximately 3.1 kb) or AAV-CSF1 (approximately 2.3 kb). Both vectors utilize the CAG promoter to enhance gene expression efficiency. The CAG promoter exhibits high expression stability in the nervous system, avoiding common epigenetic silencing issues, ensuring long-term expression and effective immune regulation, and facilitating the study of the effects of long-term immune microenvironmental manipulation. Regarding serotype selection, AAV9 or AAV-PHP.eB is recommended to penetrate the blood-brain barrier and achieve high transduction efficiency of glioma cells. The injection dose is 1-5E+11 vg / mouse, diluted in 100 μL of formulation buffer.

[0071] Here, AAV vectors have enhanced blood-brain barrier penetration (particularly AAV9 and AAV-PHP.eB serotypes), can stably and sustainably express gene products, and have low immunogenicity in small animals. This technology improves gene delivery efficiency, reduces off-target effects, and can more effectively modulate the tumor immune microenvironment.

[0072] S7, AAV transgene expression monitoring

[0073] To monitor AAV transgene expression, various methods can be used. mRNA expression levels can be measured using qPCR, while protein expression levels can be measured using Western blot or ELISA. Furthermore, immunohistochemistry (IHC) or immunofluorescence (IF) staining can be used to further verify transgene expression in tissues.

[0074] S8, Immune microenvironment and tumor growth monitoring

[0075] After establishing the immune microenvironment, the immune cells in the tumor are analyzed by flow cytometry, especially the infiltration of immune cells such as CD8+ T cells, Tregs, MDSCs, and macrophages. Compared with existing glioma mouse models, which can usually only be evaluated in terms of tumor growth, this application can monitor the immune cell composition and changes of the tumor microenvironment in real time, helping to deeply understand the immune escape mechanism and the dynamic changes of the immune microenvironment, and providing a more sophisticated research tool for the evaluation and optimization of immunotherapy.

[0076] Immunofluorescence staining is also used to detect the expression of immune co-stimulatory / co-inhibitory molecules (such as PD-L1, CD80 / CD86, etc.). In addition, cytokine levels (such as IL-6, IFN-γ, etc.) can also be detected using ELISA or Luminex. Tumor growth is monitored using small animal MRI or CT scans to assess changes in tumor volume and to observe tumor cell morphology using HE staining. Compared to traditional tumor models, the use of non-invasive imaging technology allows for real-time and continuous monitoring of tumor growth, reducing harm to experimental animals, improving experimental efficiency, and increasing data reproducibility.

[0077] S9, Validation and Advantages of the Model

[0078] The reproducibility of the model was verified by statistically analyzing tumor incidence and growth rates across different experimental batches. Furthermore, the model was compared with other existing glioma models to assess its response to immunotherapy or targeted therapies. Finally, the scientific validity and clinical translation potential of the model were evaluated using metrics such as survival time, immune infiltration, and gene expression profiles.

[0079] The above steps are described in detail below with reference to several groups of embodiments:

[0080] Example 1: Validation of immune reconstitution of human CD34+ mesenchymal stem cells in NOG mice

[0081] Experimental animals and groups

[0082] In this study, 3-week-old female NOG mice (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicCrl) was purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0083] The irradiated NOG mice were randomly divided into two groups according to the experimental requirements, with 5 mice in each group. In the blank control group, each mouse was injected with 100 μL PBS into the tail vein; in the stem cell immune reconstitution group, each mouse was injected with Human CD34+ mesenchymal stem cells (suspended in 100 μL PBS).

[0084] Immune reconstitution monitoring

[0085] Peripheral blood was collected from mice approximately 4 weeks after transplantation with human CD34+ mesenchymal stem cells (50-100 μL / time, EDTA anticoagulation). Immune reconstitution was verified by flow cytometry using a flow cytometry antibody panel (all purchased from BioLegend). Detailed information is available at: Figure 8 shown.

[0086] Flow cytometry operation

[0087] Erythrocyte lysis: Incubate with ACK lysis buffer (Thermo Fisher) at room temperature for 3-5 minutes; centrifuge at 300 × g for 5 minutes at room temperature to collect white blood cells; aspirate the supernatant, leaving approximately 50 µL of liquid to avoid disturbing the pellet; gently mix the cells and remaining liquid, then add 5 mL of pre-chilled PBS buffer; mix the cells and buffer, then collect the cells by centrifugation at 300 × g for 5 minutes at 2-8°C; aspirate the supernatant, and then resuspend the cells in PBS buffer added as needed at 2-8°C.

[0088] Antibody staining: Add antibodies as needed and as specified in the antibody instructions, incubate for 20 minutes (4°C) in the dark; resuspend and dilute in approximately 1 mL of PBS, centrifuge at 300 × g for 5 minutes to collect cells; resuspend in 100 µL of FACS buffer and load onto the microscope.

[0089] Data analysis: BD FACSDiva software was used for acquisition and FlowJo v10.8 for analysis.

[0090] The experimental results and schematic diagram are shown in Figure 2 and Figure 9 As shown, Figure A: FSC / SSC population diagram of total cells, showing granulocytes (high FSC / SSC), monocytes (medium FSC / SSC) and lymphocytes (low FSC / SSC); Figure B: CD45+ cell sorting (abscissa CD45 PerCP-Cy5.5, ordinate FSC); Figure C: CD3+ and CD3- cell populations (abscissa CD3 APC, ordinate CD45PerCP-Cy5.5); Figure D: CD4+ Th and CD8+ Tc subset analysis (abscissa CD4 BV421, ordinate CD8a PE-Cy7); Figure E: CD19+ B cell and CD56+ NK cell populations (abscissa CD19 FITC, ordinate CD56 PE).

[0091] Experimental Conclusion

[0092] This example demonstrates that CD34+ MSCs transplantation can significantly reconstitute human T cells (CD3+) and B cells (CD19+) by flow cytometry. Figures AE clearly demonstrate the cell population and subpopulation ratios, demonstrating that the method described in the present invention can effectively establish the immune basis of a humanized glioma PDX model.

[0093] Example 2: Effects of different immune reconstitution conditions on the intracranial growth curve of mice after brain glioma tissue cell transplantation

[0094] Experimental animals and groups

[0095] In this study, 3-week-old female NOG mice (NOD.Cg- Prkdc scid Il2rg tm1Sug / JicCrl) was purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0096] The irradiated NOG mice were randomly divided into 3 groups according to the experimental requirements, with 5 mice in each group. The blank control group was injected with 100 μL PBS into the tail vein of each mouse; the stem cell immune reconstitution group was injected with 100 μL PBS into the tail vein of each mouse. Human CD34+ mesenchymal stem cells (suspended in 100 μL PBS) were injected into the tail vein of the non-immune intervention group. Mouse embryonic fibroblasts NIH3T3 (suspended in 100 μL PBS).

[0097] Immune reconstitution monitoring

[0098] Peripheral blood (50-100 μL / time, EDTA anticoagulation) was collected from mice approximately 4 weeks after transplantation of human CD34+ mesenchymal stem cells. Immune reconstitution was verified by flow cytometry. The flow cytometry antibody panel (all purchased from BioLegend) and specific antibody information and flow cytometry detection process were the same as in Example 1.

[0099] Tumor transplantation after immune reconstitution

[0100] Patient-derived tumor tissue that meets the screening criteria (pathologically confirmed as IDH wild-type glioblastoma) was digested into a cell suspension and implanted into the left or right striatum of mice (coordinates: 2.0 mm posterior to the anterior bregma, 2.0 mm left or right of the midline, depth 2.5-3.0 mm). The specific digestion process is as follows:

[0101] Single cell suspension preparation-mixed enzyme digestion method

[0102] a) After all tumors are removed, transfer the tumors to the pretreatment solution and use curved scissors to cut the tumors into 1-3 mm. 3 Let the pieces stand for a few seconds, use a 1 mL pipette to aspirate the smaller particles on the upper layer, continue to mince the tissue, and repeatedly add physiological saline until all tissues meet the requirements. This process should be carried out on ice for no more than 30 minutes.

[0103] b) Transfer the tumor tissue suspension to a 50 mL centrifuge tube, add Neurobasal medium, centrifuge at 250 × g for 5 minutes, discard the supernatant, and add 20 mL of tumor tissue digestion solution (Neurobasal medium, final concentrations of 0.1% collagenase IV, 0.125% trypsin, and 0.002% DNase).

[0104] c) Mix thoroughly by gently pipetting. Transfer the culture to a culture flask and incubate on a shaker at 37°C for 1-2 hours. Observe the digestion status under a microscope every 30 minutes to determine whether to terminate the digestion.

[0105] d) After digestion, dilute with PBS and remove any remaining tissue fragments using a 200-mesh sieve. Wash twice with 5-10 volumes of PBS buffer and filter until no tissue fragments remain to obtain a single-cell suspension.

[0106] e) Collect the single-cell suspension, centrifuge at 300 × g for 5 minutes, and discard the supernatant.

[0107] f) Resuspend the cells in PBS (with one-tenth volume of Matrigel added) and adjust the cell concentration to 5 × 10 8 / mL.

[0108] Tumor growth monitoring

[0109] After the animal model of brain glioma was established, the present invention used a small animal MRI imaging system to monitor the tumor growth dynamics, as follows:

[0110] T2-weighted fast spin echo (T2WI-TSE) scans were performed using a Bruker BioSpec 70 / 30 7T small animal magnetic resonance imaging system with the following scan parameters: TR / TE = 3000 / 50 ms, slice thickness 0.5 mm, field of view 20 × 20 mm, matrix 256 × 256, and NEX = 2. Scans were performed regularly after orthotopic tumor implantation.

[0111] Tumor volume analysis was performed using Paravision 6.0 software. The specific steps were as follows: importing the acquired images into the software, identifying high-signal tumor areas, delineating the ROI layer by layer, and using the system's built-in volume measurement module to automatically calculate the tumor volume based on the ROI area and thickness of each layer. The data that changes over time were then exported to determine the growth of the tumor in the model and the effectiveness of the establishment of immune regulatory effects. The specific results are as follows.

[0112] The experimental results are as follows Figure 3 and Figure 10 The tumor volume of each model construction group increased over time, indicating that the model construction was in line with expectations. The tumor volume of the PBS control group and the non-immune intervention group (NIH3T3) increased significantly faster than that of the stem cell injection group ( P <0.01), but there was no significant difference between the PBS control group and the non-immune intervention group (NIH3T3), indicating that non-human cells cannot effectively regulate the tumor microenvironment; after the human immune system was reconstructed, the tumor growth itself was inhibited to a certain extent, reflecting the importance of constructing the immune microenvironment.

[0113] Example 3: Target protein after AAV injection TGF-β1 、 IL10 、 CSF1 mRNA expression in peripheral blood and tumor tissue

[0114] This example is used to verify the expression effect of the constructed AAV-mediated expression system in brain glioma model mice, and to detect TGF-β1 、 IL10 、 CSF1 mRNA levels of three immune regulatory factors.

[0115] The experimental animals were NOG mice whose immune systems had been reconstituted with human CD34+ hematopoietic stem cells and orthotopically inoculated with glioma donor tumor cells. After modeling, the animals were randomly divided into two groups, with 8 model mice in each group.

[0116] Control group (blank group): The same volume of preparation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20);

[0117] Experimental group (AAV immune reconstitution group): mixed AAV vectors were injected via tail vein, and the mixed AAV vectors contained:

[0118] AAV-TGFβ1-P2A-IL10 vector (co-expression of TGF-β1 and IL10 driven by the CAG promoter);

[0119] AAV-CSF1 vector (CSF1 expression driven by CAG promoter), the structure is as follows Figure 13 and Figure 14 shown.

[0120] The AAV injection dose was 1E+11 vg per mouse and dissolved in 100 μL formulation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl, and 0.005 mM Tween-20).

[0121] 3-4 weeks after injection, peripheral blood was collected from mice through the submandibular vein and PBMC cells were isolated. At the same time, some animals were sacrificed and tumor tissues were isolated.

[0122] Total RNA from peripheral blood PBMC and tumor tissue was extracted using an RNA extraction kit, and cDNA was synthesized using a reverse transcription kit, and then detected using qPCR. TGF-β1 、 IL10 and CSF1 The mRNA expression level of GAPDH as an internal reference gene.

[0123] The experimental results showed that compared with the control group, the AAV injection group had a significantly higher expression of PBMC in peripheral blood and tumor tissue. TGF-β1 、 IL10 and CSF1 The mRNA expression levels of genes were significantly upregulated ( P <0.01), indicating that the constructed AAV vector can effectively express the target immune regulatory factors in vivo and has the functional basis for mediating the regulation of the immune microenvironment of brain glioma. The specific results are as follows Figure 4 and Figure 5 shown.

[0124] Example 4: Immunohistochemistry to determine the expression of target proteins TGF-β1, IL10, and CSF1 in tumor tissue after AAV injection

[0125] To further verify whether the constructed AAV vector can successfully express the target protein related to immune regulation in vivo, immunohistochemical staining was used to detect the expression of TGF-β1, IL10 and CSF1 in glioma tissues.

[0126] The experimental animals were NOG mice whose immune systems had been reconstituted with human CD34+ hematopoietic stem cells and orthotopically inoculated with glioma donor tumor cells. After modeling, the animals were randomly divided into two groups, with eight model mice in each group.

[0127] Control group (blank group): The same volume of preparation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl and 0.005 mM Tween-20. );

[0128] Experimental group (AAV immune reconstitution group): mixed AAV vectors were injected via tail vein, and the mixed AAV vectors contained:

[0129] AAV-TGFβ1-P2A-IL10 vector (co-expression of TGFβ1 and IL10 driven by the CAG promoter);

[0130] AAV-CSF1 vector (CSF1 expression driven by CAG promoter), the structure is as follows Figure 13 and Figure 14 shown.

[0131] The AAV injection dose was 1 × 10¹¹ vg per mouse, dissolved in 100 μL formulation buffer (containing 20 mMTris, 1 mM , 200 mM NaCl, and 0.005 mM Tween-20).

[0132] Three to four weeks after injection, some animals were sacrificed and brain glioma tissues were isolated and fixed in 4% paraformaldehyde for 24 hours. After routine dehydration and embedding, paraffin sections (slice thickness 4 μm) were prepared for subsequent immunohistochemical staining.

[0133] The dyeing process is as follows:

[0134] The sections were dewaxed with xylene and rehydrated with gradient ethanol, and then antigen retrieval was performed using citrate buffer;

[0135] Use 3% Block endogenous peroxidase activity;

[0136] Normal goat serum was used to block nonspecific binding sites;

[0137] According to the experimental design, the primary antibodies were added to the tissue sections: rabbit anti-mouse TGF-β1, IL10, and CSF1 antibodies, and incubated at 4°C overnight;

[0138] The next day, HRP-labeled goat anti-rabbit secondary antibody was added and incubated at room temperature for 1 hour;

[0139] DAB color development, hematoxylin counterstaining, dehydration and sealing;

[0140] Observe under a light microscope and take photos.

[0141] like Figure 6 The results showed that the expression of TGF-β1, IL10 and CSF1 proteins in the tumor tissues of mice in the AAV injection group was significantly enhanced, with brown-yellow particles mainly distributed in the tumor cells and the surrounding infiltrating immune cells. Compared with the control group, the expression levels were significantly increased ( P <0.01).

[0142] This result is consistent with the mRNA detection result, further verifying that the AAV-TGFβ1-P2A-IL10 vector and the AAV-CSF1 vector can synergistically drive the expression of target immune regulatory factors in vivo, thereby laying the foundation for regulating the tumor immune microenvironment.

[0143] Example 5: Flow cytometry determination of changes in the immune microenvironment of tumor tissue after immune system reconstitution

[0144] This example aims to compare the differences in the composition of the immune microenvironment in glioma tissue before and after stem cell immune reconstruction and AAV-mediated immune regulation by flow cytometry, thereby verifying the effect of the AAV-mediated immune microenvironment construction method of the present invention on the tumor immune status.

[0145] Three-week-old female NOG mice were selected for the experiment. The mice were randomly divided into three groups, with six mice in each group. According to the experimental design, the mice in each group were operated according to the following plan.

[0146] Single glioma model control group: no immune reconstruction was performed, only the glioma model was established;

[0147] Stem cell reconstruction + glioma model group: received CD34+ stem cell reconstruction and established glioma model after reconstruction;

[0148] Stem cell + AAV + glioma model group: received CD34 + stem cell reconstruction, and after reconstruction, the glioma model was established and the AAV vector mixture was injected through the tail vein to further construct an immune regulatory microenvironment.

[0149] The hybrid AAV vector comprises:

[0150] AAV-TGFβ1-P2A-IL10 vector (co-expression of TGF-β1 and IL10 driven by the CAG promoter);

[0151] AAV-CSF1 vector (CSF1 expression driven by CAG promoter), the structure is as follows Figure 13 and Figure 14 shown.

[0152] The AAV injection dose was 1E+11 vg per mouse and dissolved in 100 μL formulation buffer (containing 20 mM Tris, 1 mM , 200 mM NaCl, and 0.005 mM Tween-20).

[0153] Four weeks after AAV injection (i.e., five weeks after tumor inoculation), mice were sacrificed, tumor tissue was isolated, and single-cell suspensions were prepared by enzymatic hydrolysis. The hydrolysis solution consisted of RPMI-1640 medium containing 1 mg / mL collagenase IV and 0.1 mg / mL DNase I. After incubation in a 37°C water bath for 30 minutes, the cells were filtered through a 70 μm cell sieve, erythrocytes were removed using ACK lysis buffer, and the cells were resuspended in PBS for subsequent staining. Surface staining was performed using human-specific antibodies, including:

[0154] T cell subsets: ; Myeloid cells: ; Immunosuppressive cells: type macrophages, ; Cytokine-related markers: , cell;

[0155] Use Figure 11 Human antibodies were used for surface and intracellular staining, and all antibodies were purchased from Biolegend or BD.

[0156] After staining, resuspend in FACS buffer for detection.

[0157] The data were acquired using BD FACSDiva software and analyzed using FlowJo v10.8.

[0158] The main results are as follows Figure 12 As shown:

[0159] Treg cells in tumor tissue in the combined reconstruction group The proportion increased significantly; accompanied by M2 macrophages increase; and The overall cell ratio decreased slightly compared with the stem cell reconstruction group; and The cells were significantly increased in the combined reconstruction group; there was an increasing trend of the above factors in the simple AAV intervention group, but lower than the combined group; the levels of the above immunosuppressive cells / factors were the lowest in the simple stem cell reconstruction group.

[0160] in conclusion:

[0161] The results of this example show that although simple stem cell reconstruction can partially restore the infiltration of human immune cells, it has not yet formed a tumor-related immune microenvironment; and further using the AAV-mediated method of the present invention on the basis of stem cell reconstruction can effectively induce the expression of immune regulatory factors such as TGF-β1, IL10, and CSF1, significantly changing the types and proportions of immune cells in the tumor microenvironment, tending to the enrichment of M2 macrophages and the upregulation of regulatory T cells, presenting the characteristics of an "immunosuppressive microenvironment", verifying the effectiveness and feasibility of the present invention in constructing an immune microenvironment.

[0162] In summary, the present invention achieves significant improvements in the following aspects:

[0163] Accurately simulate the immune microenvironment of human GBM.

[0164] Traditional models have failed to effectively reproduce the immunosuppressive characteristics of GBM. However, this approach uses AAV-mediated immune regulation (such as key immune factors such as PD-L1, CSF1, and TGF-β1) to enhance Treg activity and increase M2 macrophage infiltration, making the immune environment closer to that of human GBM patients.

[0165] This strategy is more consistent with the tumor immune characteristics of GBM, making it more suitable for studying immune escape mechanisms and developing immunotherapy strategies.

[0166] A single injection of AAV can achieve long-term immune microenvironment regulation.

[0167] Traditional GBM models usually rely on local injection or gene editing to change the immune environment. However, this protocol uses a single tail vein injection of AAV to achieve long-term and stable expression of specific immune regulatory factors in the central nervous system (CNS).

[0168] This method not only reduces the stress response and surgery-related injuries of experimental animals, but also dynamically regulates the immune microenvironment throughout the disease process, making the model more physiologically relevant.

[0169] The modeling is stable and fast, which improves the experimental efficiency.

[0170] By optimizing the modeling process, the repeatability and reliability of the model can be improved, the research process can be accelerated, and the experimental efficiency can be improved.

[0171] It is more suitable for immunotherapy research and can improve clinical translation value.

[0172] Traditional GBM mouse models have poor predictive power for immunotherapy (such as PD-1 antibodies and CAR-T therapy). However, this protocol improves the research reliability of strategies such as immune checkpoint inhibitors (ICIs), CAR-T cell therapy, and tumor vaccines by optimizing the immune microenvironment.

[0173] This model better simulates the immune response characteristics of clinical GBM and can serve as an important tool for screening and optimizing immunotherapy strategies.

[0174] This protocol overcomes the limitations of existing GBM animal models, providing a more precise and efficient model for studying GBM development and its immune microenvironment. This model not only improves the accuracy of immunotherapy research but also accelerates drug screening and clinical translation, providing stronger support for basic research and clinical treatment of GBM.

[0175] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing an animal model of glioma based on AAV-mediated immune microenvironment, characterized in that: The following steps are involved: Select immunodeficient mice of appropriate age; Immune system reconstitution was performed by tail vein injection of human CD34+ hematopoietic stem cells; Flow cytometry was used to detect human CD45+ immune cells in peripheral blood PBMCs to confirm the successful reconstitution of the immune system; Select tumor tissues that highly express glioma marker genes and perform rapid qPCR or targeted RNA-seq immunohistochemistry multiplex validation to screen tumor tissues that are consistent with glioma animal models; The selected tumor tissues were physically cut into pieces and treated with a pretreatment solution before being digested into single-cell suspensions, which were then inoculated into the mouse skull using stereotactic injection. Injecting an AAV vector carrying a specific immune regulatory gene into the tail vein of mice to regulate the immune microenvironment of brain gliomas. When stable expression of the AAV expression product is detected in the mice, the immune regulation mediated by the AAV expression product is completed. When stable tumor tissue growth and immune cell infiltration characteristics are observed, it is confirmed that the immune microenvironment reconstruction and tumor growth meet the requirements of model construction, and the construction of the brain glioma animal model is completed; The AAV vector includes an AAV-TGFβ1-P2A-IL10 expression cassette and an AAV-CSF1 expression cassette for expressing TGF-β1, IL10 and CSF1 genes; The AAV-TGFβ1-P2A-IL10 expression cassette includes a nucleic acid structure for co-expression of the TGF-β1 gene driven by a CAG promoter and the IL10 gene through a P2A self-cleavage peptide sequence, and the total length of the nucleic acid sequence is 3.1±0.2 kb; The AAV-CSF1 expression cassette includes a CSF1 gene expression unit driven by a CAG promoter, and the total length of the nucleic acid sequence is 2.3±0.2 kb.

2. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: AAV9 or AAV-PHP.eB serotypes were used for delivery of TGF-β1, IL10, and CSF1 genes.

3. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The injection dose of the AAV vector is 1-5E+11 vg / mouse; The AAV vector was formulated in a buffer containing 20 mM Tris, 1 mM MgCl2, 200 mM NaCl, and 0.005 mM Tween-20 at a pH of 7.7 to 8.

3.

4. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The brain glioma marker genes and their key molecular changes include: Upregulation of CD133 protein expression, R132H mutation of IDH1 gene, amplification of EGFR gene, methylation of MGMT gene promoter and upregulation of SOX2 gene expression were observed.

5. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The pretreatment solution includes Neurobasal culture medium and Matrigel mixed at a volume ratio of 3:1, and supplemented with 2×B27, 2 mM glutathione, 50 U / mL penicillin-streptomycin, and 20 ng / mL PDGF-BB.

6. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The growth monitoring of the tumor tissue is obtained by combining T2-weighted images of small animal MRI with volume change calculation.

7. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The cell morphology of the tumor tissue was observed by HE staining.

8. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: Methods for detecting significant expression of AAV expression products in mice, comprising: The expression level of mRNA was detected by qPCR technology; Western Blot or ELISA techniques were used to detect protein expression levels; Immunohistochemical staining or immunofluorescence staining was used to further verify the transgene expression product in the tissue.

9. The method for constructing an animal model of glioma based on AAV-mediated immune microenvironment according to claim 1, characterized in that: The tumor tissue is selected from tumor tissue that is pathologically confirmed to be WHO grade III or IV.

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