Application of Semaphorin 3G overexpression adeno-associated virus vector in preparation of brain glioma treatment medicine

Abstract

The invention belongs to the field of tumor drugs, and particularly relates to application of an adeno-associated virus vector overexpressing Semaphorin 3G in preparation of drugs for treating brain glioma. The AAV-SEMA3G can effectively reduce the growth speed of brain glioma of mice and prolong the survival time of tumor-bearing mice, and can be particularly used for preparing drugs for treating brain glioma and the like. The invention discloses an application of AAV-SEMA3G in preparation of a medicine for treating brain glioma.

Description

Technical Field

[0001] The present invention belongs to the field of tumor drugs, and particularly relates to the use of adeno-associated virus AAV-SEMA3G that overexpresses Semaphorin 3G in the preparation of drugs for treating brain gliomas. Background Art

[0002] Malignant glioma is the most common and most aggressive primary brain tumor in adults, with an incidence of approximately 3.1 per 100,000 people. However, glioblastoma accounts for approximately 45.6% of all primary malignant brain tumors, and its incidence increases with age. Glioblastoma has a poor prognosis and limited treatment options. The main treatment for glioblastoma is surgical resection of the tumor. Although maximal resection can provide patients with better progression-free and overall survival, surgical resection cannot cure the disease. Therefore, further adjuvant treatment with radiotherapy and chemotherapy is required. Although temozolomide chemotherapy and radiotherapy can improve patient survival, tumor progression or recurrence will eventually occur.

[0003] Adeno-associated virus (AAV) vectors have shown unique potential in the treatment of gliomas. Their low immunogenicity, long-term gene expression capacity, and high biosafety make them ideal tools for the targeted delivery of therapeutic genes. In malignant gliomas, AAV can achieve tumor-specific targeting by modifying the capsid protein or promoter, delivering tumor suppressor genes (such as p53), suicide genes (such as HSV-TK), or immunomodulatory factors (such as IL-12) to the tumor, inducing tumor cell apoptosis or activating anti-tumor immune responses. In addition, AAV combined with gene editing technologies such as CRISPR-Cas9 can precisely knock out oncogenic mutations (such as EGFRvIII) or repair tumor suppressor genes. Recent studies have also taken advantage of the ability of AAV to cross the blood-brain barrier, achieving central delivery through local intracranial injection or systemic administration, and enhancing chemoradiotherapy sensitivity by regulating the tumor microenvironment (such as inhibiting angiogenesis or remodeling immunosuppressive cells). Despite challenges such as limited vector capacity and transfection efficiency restricted by serotype, the innovative application of AAV in glioma gene therapy, oncolytic virus combination therapy and personalized precision medicine is driving the progress of clinical trials and providing new strategies for this difficult-to-treat tumor.

[0004] Semaphorin 3G (SEMA3G) is a secreted protein derived from vascular endothelial cells and a member of the third semaphorin subfamily, first discovered in 2005. A recent study demonstrated that SEMA3G degrades pathological neovascularization and promotes the formation of normal vascular networks. Furthermore, SEMA3G inhibits the migration of glioma U251 cells by reducing the expression of matrix metalloproteinase 2 (MMP2). SEMA3G exhibits significant anti-angiogenic effects in breast cancer cells MDA-MB-231 and has also been found to significantly inhibit the growth of MDA-MB-435 cells.

[0005] The present invention discovered that adeno-associated virus AAV-SEMA3G overexpressing Semaphorin 3G blocked the growth rate of glioma tissue derived from glioma stem cells and increased the survival time of tumor-bearing mice. Summary of the Invention

[0006] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide an application of an adeno-associated virus vector overexpressing Semaphorin 3G in the preparation of a drug for treating brain glioma.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] Application of adeno-associated virus AAV-SEMA3G overexpressing Semaphorin 3G in the preparation of gene therapy drugs for brain glioma.

[0009] As a preferred embodiment of the present invention, the adeno-associated virus vector used is AAV2 / 9.

[0010] As a preferred embodiment of the present invention, the drug exerts anti-glioma activity by inhibiting glioma stem cells.

[0011] As a preferred embodiment of the present invention, the glioma includes low-grade glioma, glioblastoma, astrocytoma, and oligodendroglioma.

[0012] Specifically:

[0013] 1. Detection of SEMA3G Expression in Glioma Patients: Tumor tissues were collected from glioma patients, and SEMA3G expression levels were detected using real-time fluorescence quantitative PCR. The results showed that SEMA3G mRNA levels in glioma tissues were significantly reduced. These data suggest that SEMA3G plays an important role in the pathological progression of glioma.

[0014] 2AAV-SEMA3G mediates increased SEMA3G levels in the local microenvironment: SEMA3G overexpression AAV virus was injected into the brain of nude mice, causing high expression of SEMA3G in local brain tissue.

[0015] Effect of 3AAV-SEMA3G on Mouse Glioma Growth: Glioma stem cells (GSC07) were orthotopically inoculated into the brains of nude mice overexpressing SEMA3G to establish a mouse glioma model. The results showed that the SEMA3G overexpression group significantly slowed glioma growth, reduced tumor size, and increased mouse survival.

[0016] Beneficial Effects: This invention, through adeno-associated virus-mediated localized overexpression of SEMA3G protein, ameliorates the pathological progression of glioma-bearing mice, inhibits tumor growth, and improves mouse survival. The research findings of this invention will provide drug targets and candidate protein drugs for the development of drugs related to glioma and other tumor stem cells, as well as gene therapy strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the expression level of SEMA3G in tumor tissues of glioma patients;

[0018] Figure 2 is the AAV-SEMA3G map and overexpression efficiency;

[0019] Figure 3 The growth rate of mouse glioma after AAV-SEMA3G injection;

[0020] Figure 4 Survival time of tumor-bearing mice after AAV-SEMA3G injection. DETAILED DESCRIPTION

[0021] Experimental Methods: Tumor tissue and non-tumor control tissue samples were collected from glioma patients. Real-time fluorescence quantitative PCR was used to measure SEMA3G expression levels, confirming the involvement of SEMA3G in glioma pathology. Intracranial injection of AAV-SEMA3G induced high SEMA3G expression in the brain. Using an orthotopic mouse glioma model, it was demonstrated that regulating SEMA3G protein levels affected tumor growth and survival in mice.

[0022] The adeno-associated virus AAV2 / 9-h-CMV-SEMA3G (i.e., AAV-SEMA3G) overexpressing Semaphorin 3G and the control virus AAV2 / 9-ZsGreen (i.e., AAV2-Con) in the following examples and their construction methods have been disclosed in Example 4 of CN109821009B.

[0023] Example 1 Detection of SEMA3G expression levels in glioma patients:

[0024] Real-time fluorescence quantitative PCR was used to detect the expression level of SEMA3G in patients: tumor tissues and non-tumor brain tissues from glioma patients were collected as controls (brain tissues from patients with brain trauma, 5 cases each). After RNA was extracted, it was reverse transcribed into cDNA, and then the real-time fluorescence quantitative PCR method was used to detect the expression level of SEMA3G in patients.

[0025] The test results are attached. Figure 1 The results showed that the expression level of SEMA3G in glioma tissue was significantly reduced.

[0026] Example 2 AAV-SEMA3G-mediated SEMA3G overexpression in the brain:

[0027] Adeno-associated virus-mediated SEMA3G overexpression: Five-week-old immunodeficient mice were pre-injected with AAV2 / 9-ZsGreen control virus and AAV2 / 9-h-CMV-SEMA3G overexpression virus in their striatum. Two weeks after viral expression, the mouse striatum was collected and the SEMA3G expression level was detected by QPCR and Western blot.

[0028] The test results are attached. Figure 2 , compared with the control group, the expression level of SEMA3G in the striatum of mice in the AAV-SEMA3G injection group was significantly increased.

[0029] Example 3 AAV-SEMA3G inhibits the growth rate of mouse glioma:

[0030] (1) Construction of Luciferase-stable GSC07 cell line: GSC07 cells were seeded in a 6-well plate and placed in an incubator overnight; the amount of virus was calculated based on the MOI value (50) and the virus titer and added to the culture medium. After 72 hours of culture, new culture medium was replaced with puromycin for screening; after 3 to 4 generations of screening, a stable cell line was obtained.

[0031] (2) Inducing SEMA3G overexpression in mouse brain tissue: Same as Example 2

[0032] (3) Orthotopic tumor model in mice: Two weeks after virus injection, GSC07 cells stably expressing luciferase were implanted into the brains of 5-week-old immunodeficient mice infected with AAV2 / 9-ZsGreen control virus and AAV2 / 9-h-CMV-SEMA3G overexpressing virus, respectively, to construct orthotopic glioma models. The mice were raised under the same conditions and their tumor-bearing survival was observed. Specifically, the experimental mice were anesthetized with 2% isoflurane and fixed on a small animal stereotaxic apparatus after the pain reflex disappeared. A hole was pre-made at the right midpoint between the anterior and posterior bregma of the mouse using a 1 mL syringe needle. 5×10 4 cells, insert the microsyringe needle along the pre-punched hole to 3.5mm, and stop the needle for 2 minutes; slowly and evenly inject the cell suspension into the mouse brain, and slowly remove the microsyringe after 5 minutes; disinfect the needle port with iodine, place the mouse in a warming device, and wait for the mouse to recover. During this period, pay attention to the mouse's physical signs; after the mouse wakes up, return it to the cage and continue to be raised.

[0033] (4) Small Animal Imaging Observation of Mouse Tumor Growth: Before imaging, D-luciferin potassium solution was injected intraperitoneally at a rate of 0.01 ml / g body weight. Solution preparation: Weigh 150 mg of solid, dissolve in a small amount of saline and dilute to 10 ml. Store at -20°C in the dark. After intraperitoneal administration of D-luciferin potassium solution, the mouse was anesthetized with 2% isoflurane and placed in the instrument for visible light imaging. The visible light quantification site was the brain. After measuring the fluorescence value of each mouse brain, the corresponding statistical analysis was performed.

[0034] (5) HE staining of mouse brain tissue: Mouse brain was removed by cardiac perfusion fixation: the experimental mouse was anesthetized with 2% isoflurane, and after the pain reflex disappeared, the mouse was fixed on the perfusion plate with the abdomen facing upwards; the mouse abdominal skin was carefully cut open with ophthalmic scissors, the diaphragm was removed, and the chest and abdominal cavities were exposed; the ribs were carefully cut from both sides, and the ribs were turned open and fixed with hemostatic forceps; the inferior vena cava was cut, the perfusion needle was inserted into the left ventricle, and 70 mL of pre-cooled PBS was injected at a constant speed using a peristaltic pump; after observing that the liver turned khaki, the PBS was replaced with pre-cooled 4% PFA solution, and 70 mL was continuously pumped in until the whole body muscles of the mouse were stiff; the whole brain of the mouse was carefully removed and placed in a 10 ml centrifuge tube containing 4% PFA, and placed in a refrigerator at 4°C for further fixation for 6 h; the mouse brain was treated with 30% sucrose solution at 4°C until the mouse brain lost water and settled to the bottom of the centrifuge tube. For frozen sectioning, remove the mouse brain, dry it with absorbent paper, and place it in a brain mold to remove excess tissue. Prepare a base with OCT in a cryostat precooled to -20°C and smooth it out. Place the mouse brain on the smoothed base, embed it in OCT, and wait for it to solidify. Once it has completely solidified, secure the base and adjust it to the appropriate position for sectioning. The sample should be 8 μm thick and directly attached to an adhesive slide. HE staining: Return the slides to room temperature; stain the specimens in hematoxylin for 4 minutes; rinse with running water for 5 minutes; immerse the specimens in 1% hydrochloric acid alcohol for 10 seconds; debluing with 0.6% ammonia solution for 20 seconds; rinse with running water for 2 minutes; stain the specimens in eosin solution for 4 minutes; soak in 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 minutes in sequence; rinse with running water for 5 minutes; soak the specimens in 70% ethanol, 80% ethanol, 90% ethanol, and anhydrous ethanol for 5 minutes in sequence; soak the specimens in 100% xylene II and 100% xylene I for 5 minutes in sequence; seal the slides with neutral gum; photograph and interpret the results under a microscope.

[0035] The test results are attached. Figure 3 Compared with the control group mice, the tumor volume of the AAV-SEMA3G injection group mice was significantly reduced and the tumor growth rate was slowed down.

[0036] Example 4 AAV-SEMA3G prolongs the survival of glioma mice:

[0037] The AAV-SEMA3G injection and mouse modeling methods were the same as in Examples 2 and 3. After mouse modeling, the weight and survival status of the mice were recorded every day, and the survival period of the mice was recorded.

[0038] The test results are attached. Figure 4 Compared with the control group, the survival time of mice in the AAV-SEMA3G injection group was significantly prolonged.

Claims

1. Application of adeno-associated virus (AAV)-SEMA3G overexpressing Semaphorin 3G in the preparation of gene therapy drugs for brain glioma.

2. The use according to claim 1, characterized in that The adeno-associated virus vector used was AAV2 / 9.

3. The use according to claim 1, characterized in that The drug exerts anti-glioma activity by inhibiting glioma stem cells.

4. The use according to claim 1, characterized in that The gliomas include low-grade gliomas, glioblastomas, astrocytomas, and oligodendrogliomas.

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