CN116837029A - SINV vector for expressing IL-7 and GM-CSF and application thereof in preparation of antitumor drugs - Google Patents
SINV vector for expressing IL-7 and GM-CSF and application thereof in preparation of antitumor drugs Download PDFInfo
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- CN116837029A CN116837029A CN202310061339.1A CN202310061339A CN116837029A CN 116837029 A CN116837029 A CN 116837029A CN 202310061339 A CN202310061339 A CN 202310061339A CN 116837029 A CN116837029 A CN 116837029A
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Abstract
The invention discloses an SINV vector for expressing IL-7 and GM-CSF and application thereof in preparing antitumor drugs, wherein the SINV vector takes pSINV as a framework vector, and the framework vector is connected with IL-7 and GM-CSF. The invention provides a Sindbis virus infectious vector for stably expressing IL-7 and GM-CSF genes, and Sindbis virus particles prepared by the sindbis virus infectious vector can increase the quantity of T cells, CD8+ T cells and NK cells in solid tumor cells of a U87-MG cell immunodeficiency mouse model, can play an obvious role in killing tumors on the solid tumor of the U87-MG cell immunodeficiency mouse model, and has important practical significance and wide application value for application research and basic research such as oncolytic treatment of malignant glioma.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an SINV vector for expressing IL-7 and GM-CSF and application thereof in preparation of antitumor drugs.
Background
Glioblastoma (glioblastoma multiforme, GBM) is a common brain tumor, and has the problems of drug resistance, poor prognosis of patients, short survival time and the like during treatment. GBM progresses very rapidly, and current standard treatment is synchronized radiotherapy and chemotherapy after surgery. GBM has a highly invasive growth pattern, and on the premise of not damaging brain function of the patient, the tumor cannot be completely resected by surgery, and the primary tumor is easily metastasized, eventually leading to tumor recurrence and death of the patient. The direction of selecting new drugs to develop according to the treatment bottleneck of the existing drugs is an effective way for improving the treatment effect of the new drugs, and the extract and the targeted treatment direction of natural products are ideal new drug development directions.
SINV (Sindbis virus) is a virus belonging to the genus Alphavirus (Alphavirus) of the family togaviridae, and infections in humans cause fever, burnout, headache, joint pain, and rash, and are known as "Sindbis fever. SINV is characterized by animal viruses in aspects of morphology, structure, replication, translation, pathogenesis and the like, is a mild pathogen, has high operation safety, is widely used as a model virus for researching the basic rules of animal viruses at present, is especially used for vaccine development and cancer gene therapy, and can excite effective humoral immunity and cellular immune response.
Interleukin-7 (IL-7) is a cytokine which is secreted mainly by thymocytes, bone marrow stromal cells, intestinal epithelial cells, skin keratinocytes and the like, plays an important role in normal development of the human immune system and maintenance of normal immune function, is now commonly used in combination with oncolytic viruses, and can further improve the oncolytic efficiency of oncolytic viruses.
Granulocyte-macrophage colony-stimulating factor (GM-CSF), a 23kDa single-chain glycoprotein, functions critically by binding to heterodimeric receptors on the surface of bone marrow cells, thereby activating multiple signal transduction pathways. GM-CSF not only can regulate proliferation and differentiation of granulocyte-cell line hematopoietic progenitor cells, but also can promote proliferation and differentiation of antigen presenting cells such as dendritic cells and macrophages, and effectively induce proliferation of various immune cells with tumor inhibiting effect, thereby exerting anti-tumor immune response. The GM-CSF gene therapy has remarkable anti-tumor and immunoregulatory effects in lung cancer, renal cell carcinoma, bladder cancer, melanoma and hematological malignancy.
Oncolytic virus refers to a virus that infects and lyses cancer cells but does not infect normal cells, and can be naturally produced or can be made in the laboratory by altering other viruses. Some oncolytic viruses are being investigated for the treatment of cancer, which can make chemotherapy and radiation therapy easier to kill tumor cells. In 2015, the U.S. Food and Drug Administration (FDA) approved the first oncolytic viral immunotherapy for the treatment of cancer, melanoma T-VEC. This treatment involves a herpes virus designed to express immunostimulatory GM-CSF proteins and not infect normal cells, which marks the impending oncolytic virus as a powerful tool for tumor therapy.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an SINV infectious vector for stably expressing IL-7 and GM-CSF, virus particles and application thereof, and the influence of the virus particles on tumor microenvironment and the effect in a malignant glioma U87-MG mouse subcutaneous tumor model are evaluated. The invention successfully prepares sindbis virus particles capable of stably expressing IL-7 and GM-CSF by using sindbis virus infectious vector transfected cells, and is successfully applied to oncolytic treatment of a glioblastoma U87-MG mouse model.
The invention provides an SINV infectious vector for stably expressing IL-7 and GM-CSF, wherein the SINV infectious vector takes pSINV as a framework vector, and the framework vector is connected with IL-7 and GM-CSF; the SINV infectious vector is connected with a UBC promoter, a 5'UTR, a nucleotide sequence of an NSP1 gene, a nucleotide sequence of an NSP2 gene, a nucleotide sequence of an NSP3 gene, a nucleotide sequence of an NSP4 gene, a 26s promoter, a nucleotide sequence of an IL-7 gene, a 26s promoter, a nucleotide sequence of a CAP gene, a nucleotide sequence of an E3 gene, a nucleotide sequence of an E2 gene, a nucleotide sequence of a 6K gene, a nucleotide sequence of an E1 gene, a 26s promoter, a nucleotide sequence of a GM-CSF gene and a 3' UTR.
Further, the nucleotide sequence of the SINV infectious vector is shown as SEQ ID NO. 16.
The total length of the sequence SEQ ID NO.16 is 16053bp, and the base numbers at 15941-16053 are as follows:
gtgtgttttgtgaagttttttaggcaccttttgaaatgtaatcatttgggtcaatatgtaattttcagtgttagactag taaattgtccgctaaattctggccgtttttggcttttttgttagacttaattaa。
the invention also provides a preparation method of sindbis virus particles carrying IL-7 and GM-CSF, and cells are transfected by using the SINV infectious vector.
Further, the cell is a BHK cell.
The invention also provides sindbis virus particles carrying IL-7 and GM-CSF, which are prepared by transfecting cells with the SINV infectious vector.
The invention also provides application of the Sindbis virus particle carrying IL-7 and GM-CSF in preparing a medicine for improving tumor microenvironment.
The invention also provides application of the Sindbis virus particle carrying IL-7 and GM-CSF in preparing medicines for treating glioblastoma.
Compared with the prior art, the invention achieves the following technical effects:
(1) The invention provides a Sindbis virus infectious vector for stably expressing IL-7 and GM-CSF genes, which does not need the steps of in vitro transcription and RNA transfection, and can directly transfect cells by the Sindbis virus infectious vector, thereby saving time and operation steps. The SINV infectious vector can stably express the inserted exogenous gene, and is beneficial to developing the research related to the sindbis virus infectious vector serving as a novel oncolytic virus.
(2) The invention has important value for researching the application prospect of sindbis virus as a novel oncolytic virus, and has important practical significance and wide application value for application research such as oncolytic treatment of malignant glioma and basic research (such as killing mechanism of virus in tumor cells, change of immune cells in tumor and the like).
(3) The Sindbis virus particle prepared by the invention can increase the number of T cells, CD8+ T cells and NK cells in solid tumor cells of a U87-MG cell immunodeficiency mouse model within 5 days.
(4) The Sindbis virus particle prepared by the invention can play an obvious role in killing tumors on solid tumors of a U87-MG cell immunodeficiency mouse model within 7 days.
(5) Oncolytic virus therapy utilizes genetic engineering means to modify oncolytic viruses so as to ensure that the oncolytic viruses retain the replication capacity of the viruses, and the oncolytic viruses are delivered to tumor cells in a targeted manner to kill the tumor cells, thereby achieving the aim of treatment. The SINV virus particles of the invention can replicate in malignant glioma U87-MG cells, selectively infect tumor cells, and realize the killing effect on tumors in an immunodeficiency mouse model.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a schematic construction diagram (A) of the sindbis virus infectious vector expressing IL-7 and GM-CSF, and a schematic construction diagram (B) of pSINV GM-CSF/IL-7 in example 1 of the present invention, wherein NSP 1-4, CAP, E3, E2, 6K and E1 are sindbis virus proteins.
FIG. 2 shows a one-step growth curve (A) of the Sindbis virus infectious vector carrying IL-7 gene and GM-CSF gene of example 2 of the present invention after infection of BHK cells with 0.01MOI, and the expression of IL-7 and GM-CSF proteins at cellular levels at different time points after infection of BHK cells with virus with 0.01MOI (B).
FIG. 3 shows the effect of Sindbis virus particles carrying IL-7 gene and GM-CSF gene on cell viability after infection of BHK cells (A) and U87-MG cells (B) with different MOI according to example 3 of the present invention.
FIG. 4 shows the oncolytic treatment of solid tumors of the immunodeficiency mouse model of example 4 carrying IL-7 gene and GM-CSF gene of the present invention, using Sindbis virus particles subcutaneously planted in U87-MG cells; wherein A is an immunodeficiency mouse model subcutaneously planted by U87-MG cells and the administration flow, and B is the tumor volume change condition of the mice in the experimental group and the control group within 14 days after the beginning of the administration.
FIG. 5 shows the numbers of intratumoral T cells (A), CD8+ T cells (B) and NK cells (C) in the experimental group (SINV GM-CSF/IL-12) and the control group (PBS) after 4 days of application of the Sindbis virus particle carrying IL-7 gene and GM-CSF gene of example 5 to solid tumors of an immunodeficiency mouse model subcutaneously planted in U87-MG cells.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.
The technical scheme of the invention is conventional in the art unless specifically stated otherwise.
The cells used in the examples were purchased from American Type Culture Collection (ATCC) and the mice used were purchased from Hunan Stokes Lemonda laboratory animals Co., ltd.
Example 1
The SINV infectious vector for stably expressing IL-7 and GM-CSF is prepared by the following steps:
pSINV GM-CSF/IL-7 initiates transcription and translation of viral structural proteins and nonstructural proteins by the UBC promoter. The fragments shown in the GM-CSF gene and the SEQ ID NO.5 are respectively amplified by using primers shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and then fused into the fragment shown in the SEQ ID NO. 6;
the PCR reaction system was 50. Mu.l: 5 x ReactionBuffer:10 μl,10mM dNTPs:1 μl,10 μM Forward Primer:2.5 μl,10 μM Reverse Primer:2.5 μl, template DNA:0.5 μl, DNAPolymerase:0.5 μl, nuclear-Free Water:33 μl; the amplification conditions were: 98 ℃ 60s,98 ℃ 10s,55 ℃ 15s,72 ℃ 60s,72 ℃ 10min,16 ℃ 10min,30 cycles;
the recombinant product was transformed into competent HB101 using ApaI and NotI restriction enzymes (plasmid pSINV-EGFP has been disclosed in China patent application "CN202111117573.9 Sindbis virus vector and its viral particles and application in nerve Loop"), and then the correct clone was named pSINV GM-CSF by sequencing by inserting the fragment shown in SEQ ID No.6 into pSINV-EGFP using Vazyme homologous recombination kit, culturing the clone identified as positive by PCR and extracting the plasmid.
The primers shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 and SEQ ID NO.13 are used for amplifying fragments shown in SEQ ID NO.9 and IL-7 genes and SEQ ID NO.14 respectively, and then fused into a fragment shown in SEQ ID NO. 15;
the PCR reaction system was 50. Mu.l: 5 x ReactionBuffer:10 μl,10mM dNTPs:1 μl,10 μM Forward Primer:2.5 μl,10 μM Reverse Primer:2.5 μl, template DNA:0.5 μl, DNAPolymerase:0.5 μl, nuclear-Free Water:33 μl; the amplification conditions were: 98 ℃ 60s,98 ℃ 10s,55 ℃ 15s,72 ℃ 60s,72 ℃ 10min,16 ℃ 10min,30 cycles;
the fragment shown in SEQ ID NO.15 is inserted into pSINV GM-CSF by using SapI and Pme1 enzyme digestion, then the recombinant product is transformed into competent HB101 by using Vazyme homologous recombination kit, the clone which is identified as positive by PCR is cultivated, the plasmid is extracted for sequencing, the clone which is sequenced to correct mutation is named pSINV GM-CSF/IL-7, and the nucleotide sequence of the clone is shown as SEQ ID NO. 16.
Example 2
The one-step growth curve of SINV infectious vector stably expressing IL-7 and GM-CSF after infection of cells and the expression of IL-7 and GM-CSF proteins at the cellular level after infection of cells with Sindbis virus:
after the pSINV GM-CSF/IL-7 plasmid prepared in example 1 was extracted with a plasmid extraction kit, 2. Mu.g of the pSINV GM-CSF/IL-7 plasmid was transfected into BHK cells with 4. Mu.l of lipofectamine 2000 (Thermo Fisher), 37℃at 5% (v/v) CO 2 The resulting virus was cultured in an incubator to infect BHK cells at a dose of 0.01MOI, and the cell status was observed by an inverted fluorescence microscope at various time points, and the cells appeared to have significant cytopathic effects after 48 hours. After a part of the virus supernatant collected at different time points is split-charged, a part of the virus supernatant collected at different time points is detected by a double-layer plaque method, and a one-step growth curve of infectious clone is drawn, wherein the one-step growth curve of infectious clone is shown as A in figure 2. And subpackaging the virus supernatant collected at the time point of 48 hours of infection, and preserving at-80 ℃ for later use in subsequent experiments. Through the infection of this example, SINV GM-CSF/IL-7 infectious vector-prepared SINV GM-CSF-7 infectious vector-carrying SINV GM-7 gene and sindbis virus particles were obtained. The incubation period and the cleavage amount of the Sindbis virus carrying the IL-7 gene and the GM-CSF gene and the titer of the virus at different time points can be obtained through a one-step growth curve so as to further know the growth characteristics of the modified virus.
Cell samples were collected 24 hours, 48 hours and 72 hours after infection of BHK with Sindbis virus particles carrying IL-7 gene and GM-CSF gene at 0.01MOI, and treated with 1xSDS-PAGE buffer of YEASEN. The samples were then denatured by heating them in a metal bath at 95℃for 5 minutes, after which the samples were loaded onto a 10% SDS-PAGE gel for separation, after which the proteins were transferred to PVDF membrane, blocked with 5% skimmed milk for 1 hour at room temperature and incubated overnight in a refrigerator at 4℃with Abcam's Anti-IL-7 Anti-body (cat# ab 193358) and Proteintech's GM-CSF Polyclonal antibody (cat# 17762-1-AP), respectively. The next day the PVDF membrane was washed with 1XTBST buffer, incubated with Proteintech HRP-conjugatedAffinipure GoatAnti-Rabbit IgG secondary antibody for 2 hours at room temperature, and after washing the PVDF membrane again with 1XTBST buffer, imaging of protein bands was performed, the results are shown in FIG. 2B. The appearance of protein bands suggests that sindbis virus vectors carrying the IL-7 gene and GM-CSF gene are capable of successfully expressing IL-7 protein and GM-CSF protein in cells.
Example 3
Infection of BHK and U87-MG cells with Sindbis virus particles carrying IL-7 Gene and GM-CSF Gene of the present invention, and Effect on viability of BHK and U87-MG cells:
fresh BHK and U87-MG cells were infected with the virus supernatant stored for later use in example 2 at 1MOI, 0.1MOI and 0.01MOI, respectively, after 24 hours, the supernatant was discarded, and dead living cells were identified with the Calcein/PI cell viability assay kit of Beyotime, wherein the living cells were stained with Calcein and displayed green fluorescence, and the red fluorescent label was PI-stained dead cells. Viable cells were counted in each well and cell viability was calculated for different MOI infections, the results are shown in figure 3. The viability is an important indicator reflecting the state of the cells, and the results of the cell viability test further demonstrate that sindbis virus particles carrying the IL-7 gene and GM-CSF gene are more sensitive to U87-MG cells and can cause more cell killing.
Example 4
The sindbis virus particle carrying the IL-7 gene and the GM-CSF gene is applied to the oncolytic treatment condition of the immunodeficiency mouse model solid tumor which is subcutaneously planted by U87-MG cells:
after resuspension of U87-MG cells with PBS, 100. Mu.l of the cells were taken containing 5X 10 6 Cell suspensions of individual U87-MG cells were prepared by subcutaneous injection Nu at 10 weeks of age 3-4 Nu female nude mice. On day 7 after tumor implantation, 10 mice were randomly divided into two groups, one group being the experimental group at the swelling100 microliters of 1x10 containing drug was injected on day 7, day 9 and day 11 after tumor implantation, respectively 6 SINV GM-CSF/IL-7 virus supernatant from each viral particle (collected during the transfection experiment of example 2) was injected as a control group with 100 microliters of PBS on days 7, 9, and 11, respectively, after tumor implantation, and the experimental procedure is shown as A in FIG. 4.
Tumor volume of model mice was calculated by measuring the length and width of tumor of mice daily before and after administration, tumor volume=1/2×length (mm) ×width (mm). The results of the change of the tumor volumes of mice in the experimental group and the control group after the administration (average tumor volume of 5 mice in each group) are shown as B in fig. 4, and the tumor-bearing volume of the mice in the control group is obviously larger than that of the mice in the experimental group after 14 days of the administration, which shows that the Sindbis virus particles carrying the IL-7 gene and the GM-CSF gene have obvious tumor killing effect when applied to the solid tumors of the immunodeficiency mouse model which are subcutaneously planted in U87-MG cells.
Example 5
The Sindbis virus particle carrying the IL-7 gene and the GM-CSF gene is applied to the cases of the number of T cells, CD8+T cells and NK cells in an immunodeficiency mouse model solid tumor which is subcutaneously planted by U87-MG cells:
after resuspension of U87-MG cells with PBS, 100. Mu.l of the cells were taken containing 5X 10 6 Cell suspensions of individual U87-MG cells were prepared by subcutaneous injection Nu at 6 weeks of age 3-4 Nu female nude mice. On day 7 after tumor implantation, 6 mice were randomly divided into two groups, one group was injected as an experimental group with 100 microliters of 1x10 6 SINV GM-CSF/IL-7 virus supernatant from each viral particle (collected during the transfection experiment of example 2) was used as a control to inject 100 microliters of PBS.
After 4 days of virus supernatant injection, tumor tissues of 6 mice in total of the control group and the experimental group were taken in 100 mm dishes, respectively. The tumor tissue of each mouse was treated with the following: tumor tissue was first minced with scissors after rinsing with 1x HBSS from Gibco and transferred to a 15 ml centrifuge tube, 5 ml of enzyme cocktail (1 g collagenase IV, 100 mg hyaluronidase and 20,000 units dnase in 100 ml HBSS solution, filtered through a 0.22um filter, sub-packaged and stored at-20 ℃) was added to the centrifuge tube and digested overnight at room temperature. Digestion was then stopped by adding Gibco RPMI 1640 medium containing serum and centrifuging for 5 min at 300g, then washing the pellet with PBS to remove the medium, re-suspending the pellet in 100. Mu.l PBS after two repeated washes with PBS to make a cell suspension, adding FITC-anti-mouse CD4, APC anti-mouse CD3, PE anti-mouse CD49b, APC/Cyanine7 anti-mouse CD8a antibodies purchased from Biolegend, incubating for 20 min on ice in the absence of light, and then analyzing with a flow cytometer, the results obtained were analyzed using Flowjo software, as shown in FIG. 5. As can be seen from FIG. 5, the numbers of T cells, CD8+ T cells and NK cells in the tumor of the mice treated with SINV GM-CSF/IL-7 are far greater than those of the control group, which indicates that Sindbis virus particles carrying IL-7 gene and GM-CSF gene increase the number of immune cells in the tumor, and lay a foundation for further and efficient killing of solid tumors.
By combining the above examples, the invention provides a Sindbis virus infectious vector for stably expressing IL-7 and GM-CSF, and the infectious vector has obvious tumor killing effect when applied to glioma cells and malignant glioma subcutaneous models; the method has wide application value in the aspects of establishment of a drug screening platform, establishment of an animal model, analysis of action mechanisms of drug killing glioma cells, analysis of mechanisms of viruses for stimulating immune responses in solid tumors, and the like.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. An SINV infectious vector for stably expressing IL-7 and GM-CSF, which is characterized in that the SINV infectious vector takes pSINV as a framework vector, and IL-7 and GM-CSF are connected to the framework vector;
the SINV infectious vector is connected with a UBC promoter, a 5'UTR, a nucleotide sequence of an NSP1 gene, a nucleotide sequence of an NSP2 gene, a nucleotide sequence of an NSP3 gene, a nucleotide sequence of an NSP4 gene, a 26s promoter, a nucleotide sequence of an IL-7 gene, a 26s promoter, a nucleotide sequence of a CAP gene, a nucleotide sequence of an E3 gene, a nucleotide sequence of an E2 gene, a nucleotide sequence of a 6K gene, a nucleotide sequence of an E1 gene, a 26s promoter, a nucleotide sequence of a GM-CSF gene and a 3' UTR.
2. The SINV infectious vector stably expressing IL-7 and GM-CSF of claim 1 wherein the nucleotide sequence of the SINV infectious vector is set forth in SEQ ID No. 16.
3. A method for preparing sindbis virus particles carrying IL-7 and GM-CSF, characterized in that cells are transfected with the SINV infectious vector of claim 1.
4. The method of producing sindbis virus particles carrying IL-7 and GM-CSF according to claim 3, wherein the cells are BHK cells.
5. Sindbis virus particles carrying IL-7 and GM-CSF, prepared by the process of claim 3.
6. Use of sindbis virus particles carrying IL-7 and GM-CSF according to claim 5 for the preparation of a medicament for improving tumor microenvironment.
7. Use of sindbis virus particles carrying IL-7 and GM-CSF according to claim 5 for the preparation of a medicament for the treatment of glioblastoma.
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