Oncolytic adenovirus construction method and application of shRNA (short hairpin ribonucleic acid) based on adenovirus-loaded c-Rel gene
By loading the c-Rel gene in the oncolytic adenovirus, the problem of strong immunosuppression of the tumor microenvironment is solved, and more effective anti-tumor treatment effects are achieved.
Patent Information
- Application Number
- CN202311627033.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-29
- Publication Date
- 2025-05-30
AI Technical Summary
The tumor microenvironment has strong immunosuppressive properties, limiting the anti-tumor efficacy of oncolytic viruses.
The shRNA sequence of the c-Rel gene was designed and constructed through the pDC315 plasmid, hTERT promoter, and U6 promoter to obtain a shuttle plasmid and packaged through adenovirus to obtain an oncolytic adenovirus loading the shRNA of the c-Rel gene.
By knocking down the expression of c-Rel gene in tumor-associated myeloid cells, the immunosuppressive microenvironment is changed and the body's anti-tumor immune response is remodeled, thereby improving anti-tumor efficacy.
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Figure CN120060367A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of oncolytic adenovirus construction, and in particular to a method for constructing an oncolytic adenovirus based on an adenovirus carrying shRNA of the c-Rel gene and its application. Background Art
[0002] According to the latest statistical data of the International Agency for Research on Cancer, cancer has replaced cardiovascular disease as the leading cause of death in high- and middle-income countries, seriously threatening people's lives and health, and the prevention and control situation is extremely severe. Although anti-tumor immunotherapy targeting immune checkpoints (such as PD1 / PDL1) has brought a revolution to cancer treatment, only a very small number of patients respond to this treatment. Therefore, exploring more effective treatment means is urgently needed at present.
[0003] In recent years, oncolytic virus therapy has developed rapidly, and more than 100 oncolytic virus therapy drugs have entered the clinical research stage. At present, the most widely used oncolytic virus in clinical experiments is oncolytic adenovirus. As early as 2006, China approved the listing of the world's first oncolytic adenovirus drug, H101. One of the main construction strategies of oncolytic adenovirus is to replace the natural viral promoter with a tissue-specific promoter to control the expression of the viral replication promoter gene, resulting in a transcriptionally regulated oncolytic adenovirus. In theory, oncolytic adenovirus replicates in tumor cells, releases the virus, and further infects other surrounding tumor cells, and ultimately can achieve the purpose of eliminating tumors. However, in fact, the efficacy of oncolytic adenovirus is restricted by many factors. For example, adenovirus type 5 antibodies are commonly present in the human body, which can clear oncolytic adenovirus, and the efficiency of tumor-specific promoters that initiate virus replication fails to reach the expected effect. In order to further improve the anti-tumor efficacy of oncolytic adenovirus, modifying oncolytic virus can significantly improve the efficacy of oncolytic adenovirus. For example, carrying a suicide gene can improve the killing efficiency of tumor cells; by carrying immune genes, the immune status of the body and the tumor local area can be regulated; by carrying anti-angiogenic genes, the activation of signals related to tumor cell metastasis can be blocked. Therefore, the combined application of oncolytic adenovirus and therapeutic genes has become an important direction and research hotspot of oncolytic virus.
[0004] c-Rel plays a decisive role in guiding the differentiation of myeloid progenitor cells into myeloid-derived suppressor cells (MDSCs) and the immune suppression function of MDSCs. In bone marrow cells, the lack of c-Rel significantly inhibits tumor growth in mice, and the c-Rel small molecule inhibitor R96 independently developed by the research group can specifically prevent the development of MDSCs, inhibit tumor growth in mice, and can significantly enhance the anti-tumor treatment of PD-1 antibody. Therefore, c-Rel has been proven to be a bone marrow checkpoint for cancer immunotherapy.
[0005] Oncolytic adenoviruses are a class of adenoviruses that have been genetically engineered to selectively replicate and kill tumor cells within tumor cells without affecting normal cells. Multiple oncolytic adenoviruses have been used in preclinical and clinical studies, and the efficacy and safety of oncolytic adenovirus type 5 have been confirmed. Currently, the clinically approved oncolytic adenovirus H101 has deleted the E1B-55kD and E3-19kD gene fragments in human adenovirus type 5. p53 is a tumor suppressor gene. The oncolytic adenovirus H101 lacking the viral E1B55KD gene enables H101 to selectively infect and kill tumor cells lacking p53 but not normal cells. However, H101 is only effective in tumors with p53 defects, and only nearly 50% of human tumors have p53 mutations, and the simultaneous deletion of E1B55KD leads to a decrease in viral replication ability and anti-tumor effect. Telomerase is a ribonucleoprotein enzyme with reverse transcriptase activity. Human telomerase reverse transcriptase (hTERT), as the rate-limiting component of telomerase, plays a decisive role in the activation of telomerase. The activity of telomerase is inhibited in most normal human cells, but high expression of hTERT and activation of telomerase can be observed in more than 85% of human malignant tumors, so cancer cells have acquired the ability of unlimited proliferation. Therefore, telomerase is considered an ideal calibrator for the tumor specificity of oncolytic adenoviruses.
[0006] Currently, due to the strong immunosuppression of the tumor microenvironment, the anti-tumor efficacy of oncolytic viruses is greatly restricted. Summary of the Invention
[0007] Based on this, in view of the technical problem that the strong immunosuppression of the tumor microenvironment in the prior art restricts the anti-tumor efficacy of oncolytic viruses, a method for constructing an oncolytic adenovirus based on an adenovirus carrying shRNA of the c-Rel gene and its application are proposed.
[0008] Provide a method for constructing an oncolytic adenovirus based on an adenovirus carrying shRNA of the c-Rel gene, the method comprising:
[0009] Design shRNA sequences of the c-Rel gene, and screen out target shRNA sequences from each of the shRNA sequences according to the silencing effect of the shRNA sequences;
[0010] Construct using the pDC315 plasmid, the hTERT promoter, the U6 promoter, and the target shRNA sequence to obtain a shuttle plasmid;
[0011] Perform adenovirus packaging according to the shuttle plasmid to obtain an oncolytic adenovirus carrying shRNA of the c-Rel gene.
[0012] Use of the oncolytic adenovirus carrying shRNA of the c-Rel gene in the preparation of a medicament for treating malignant tumors.
[0013] The method for constructing an oncolytic adenovirus carrying shRNA of the c-Rel gene based on an adenovirus proposed by the present invention designs shRNA sequences of the c-Rel gene, and screens out target shRNA sequences from each of the shRNA sequences according to the silencing effect of the shRNA sequences. Then, a shuttle plasmid is constructed through a pDC315 plasmid, an hTERT promoter, a U6 promoter, and the target shRNA sequences. Finally, adenovirus packaging is performed according to the shuttle plasmid to obtain an oncolytic adenovirus carrying shRNA of the c-Rel gene. Due to the introduction of shRNA of the myeloid immune checkpoint c-Rel gene, the oncolytic adenovirus can dissolve tumor cells, and by knocking down the expression of the c-Rel gene in tumor-associated myeloid cells, it is expected to change the immunosuppressive microenvironment and reshape the anti-tumor immune response of the body, thereby more effectively treating tumors. Brief Description of the Drawings
[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0015] Among them:
[0016] Figure 1 It is a flowchart of a method for constructing an oncolytic adenovirus carrying shRNA of the c-Rel gene based on an adenovirus in an embodiment;
[0017] Figure 2 It is an experimental data graph of an oncolytic adenovirus of a method for constructing an oncolytic adenovirus carrying shRNA of the c-Rel gene based on an adenovirus in an embodiment;
[0018] Figure 3 It is another experimental data graph of an oncolytic adenovirus of a method for constructing an oncolytic adenovirus carrying shRNA of the c-Rel gene based on an adenovirus in an embodiment;
[0019] Figure 4 It is another experimental data graph of an oncolytic adenovirus of a method for constructing an oncolytic adenovirus carrying shRNA of the c-Rel gene based on an adenovirus in an embodiment;
[0020] Figure 5Another experimental data graph of the oncolytic adenovirus constructed by the method of oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus in an embodiment;
[0021] Figure 6 Another experimental data graph of the oncolytic adenovirus constructed by the method of oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus in an embodiment. Detailed implementation manners
[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above drawings are intended to cover non-exclusive inclusion. The terms "first", "second", etc. in the specification and claims of this application or the above drawings are used to distinguish different objects and not to describe a specific order.
[0023] Reference to "embodiment" herein means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of this application. The phrase appears in various places in the specification and does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.
[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.
[0025] Please refer to Figure 2 as shown Figure 2 A flowchart showing a method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus provided in an embodiment of the present invention, including the following steps:
[0026] Step S101: Design shRNA sequences of the c-Rel gene, and screen out target shRNA sequences from each of the shRNA sequences according to the silencing effect of the shRNA sequences;
[0027] In this embodiment, multiple shRNA sequences of the c-Rel gene can be pre-designed, and the shRNA sequence with better silencing effect can be selected from each shRNA sequence of the c-Rel gene as the target shRNA sequence. As an example, the shRNA sequence of the c-Rel gene is designed and the silencing effect of the shRNA sequence of the c-Rel gene is verified, and the shRNA sequence of the c-Rel gene with a silencing efficiency greater than 70% is selected as the target shRNA sequence.
[0028] Step S102: Construct a shuttle plasmid by using the pDC315 plasmid, the hTERT promoter, the U6 promoter, and the target shRNA sequence.
[0029] In a preferred implementation manner, the original CMV promoter of the pDC315 plasmid is replaced with the hTERT promoter, and the replaced pDC315 plasmid is inserted into the U6 promoter to obtain the pDC315-hTERT-E1A-U6 plasmid; the target shRNA sequence is inserted into the pDC315-hTERT-E1A-U6 plasmid to obtain the shuttle plasmid. The human telomerase reverse transcriptase (hTERT) promoter can be used to replace the wild-type adenovirus E1A's own promoter (CMV promoter), thereby restricting the replication of the virus specifically in tumor cells, but having no effect on normal cells; secondly, the target shRNA with the ability to silence the expression of the c-Rel gene is inserted, so that while the virus lyses tumors, it further infects immunosuppressive cells in the tumor microenvironment, especially myeloid-derived immunosuppressive cells and silences the expression of their c-Rel, weakening the immunosuppressive effect of the tumor microenvironment and reshaping the body's anti-tumor immune response, so as to achieve a more effective treatment or cure of tumors.
[0030] As an example, using the AdMax adenovirus packaging system, which lacks the E1 and E3 regions of adenovirus, the hTERT-E1A fragment and the mRel shRNA fragment can be inserted into the pDC315 shuttle plasmid by enzymatic digestion and ligation respectively, transformed into Escherichia coli, and positive clones are screened in ampicillin resistance, thereby constructing the shuttle plasmid pDC315-hTERTp-E1A-EGFP-U6-mRel shRNA.
[0031] Step S103: Package adenovirus according to the shuttle plasmid to obtain an oncolytic adenovirus carrying the shRNA of the c-Rel gene.
[0032] Specifically, prepare 293 cells and viral plasmids for packaging the virus in advance, and transfect each 6-cm-diameter culture dish, the complex components of which include 1 μg of shuttle plasmid and 4 μg of pBHGlox(delta)E1,3Cre; and through the processes of virus collection, amplification, and purification, the oncolytic adenovirus Ad-shc-Rel targeting c-Rel is obtained. The oncolytic adenovirus Ad-shc-Rel targeting c-Rel is an oncolytic adenovirus carrying shRNA of the c-Rel gene. Among them, the viral plasmid includes the shuttle plasmid and the backbone plasmid.
[0033] In a preferred implementation, after obtaining the oncolytic adenovirus carrying shRNA of the c-Rel gene, in vitro knockdown verification, in vivo knockdown verification, and anti-tumor efficacy verification are carried out. Specifically, for in vitro knockdown verification, different immortalized immune cell lines including mouse melanoma cell line B16-F10 and myeloid cell line Raw264.7 are used, and the expression of c-Rel mRNA is measured after infecting with the oncolytic adenovirus. For in vivo knockdown verification and anti-tumor efficacy verification, the oncolytic virus targeting the c-Rel gene is injected intratumorally into tumor-bearing mice, and the virus is injected once every three days, and the growth trend of the mice tumors is observed. At the end of the experiment, the tumor tissues of the mice are taken, and the expression of the c-Rel gene in various cells in the tumor tissues is analyzed, especially myeloid-derived immunosuppressive cells.
[0034] The method for constructing an oncolytic adenovirus based on an adenovirus carrying shRNA of the c-Rel gene proposed in this embodiment designs the shRNA sequence of the c-Rel gene, and screens out the target shRNA sequence from each of the shRNA sequences according to the silencing effect of the shRNA sequence, and then constructs through the pDC315 plasmid, hTERT promoter, U6 promoter, and target shRNA sequence to obtain the shuttle plasmid. Finally, adenovirus packaging is carried out according to the shuttle plasmid to obtain the oncolytic adenovirus carrying shRNA of the c-Rel gene, which can, due to the introduction of shRNA of the myeloid immune checkpoint c-Rel gene, dissolve tumor cells through the oncolytic adenovirus, and knock down the expression of the c-Rel gene in tumor-associated myeloid cells in order to change the immunosuppressive microenvironment and reshape the body's anti-tumor immune response, thereby more effectively treating tumors.
[0035] In one embodiment, the shuttle plasmid is pDC315-hTERTp-E1A-EGFP-U6-mRel shRNA.
[0036] In a preferred implementation, the step of carrying out adenovirus packaging according to the shuttle plasmid to obtain the oncolytic adenovirus carrying shRNA of the c-Rel gene includes:
[0037] Step A: Use 293 cells for packaging the virus and viral plasmids to package adenovirus to obtain the oncolytic adenovirus, wherein the viral plasmids include the shuttle plasmid and the backbone plasmid.
[0038] In a preferred implementation, the backbone plasmid is pBHGlox(delta)E1,3Cre.
[0039] In a preferred implementation, the oncolytic adenovirus is Ad5-hTERTp-EGFP-mRel shRNA.
[0040] In a preferred implementation, the shRNA sequence of the c-Rel gene is the shRNA sequence of the c-Rel gene of mammals. In a preferred implementation, the mammal is a mouse, and the shRNA of the c-Rel gene of the mouse is 5'-GGATTAGTGCAGGAATCAATC-3', or 5'-GGATCTGCCGTGTGAACAAGA-3', or 5'-GGATCAACTGGAGAAGGAAGA-3'. The shRNA sequence of the c-Rel gene can also be the shRNA sequence of the c-Rel gene of humans.
[0041] In a preferred implementation, the application of the oncolytic adenovirus carrying the shRNA of the c-Rel gene in the preparation of a drug for treating malignant tumors, the drug comprising the oncolytic adenovirus vector Ad-shc-Rel of human type 5 targeting c-Rel.
[0042] In a preferred implementation, after the step of packaging adenovirus according to the shuttle plasmid to obtain the oncolytic adenovirus carrying the shRNA of the c-Rel gene, it further includes:
[0043] Step B: Replace the telomerase promoter of the oncolytic adenovirus with the promoter of E1A of human adenovirus type 5, delete the adenovirus E3 region of the oncolytic adenovirus, and insert the shRNA sequence of the c-Rel gene after the oncolytic adenovirus.
[0044] It should be noted that the oncolytic adenovirus vector Ad-shc-Rel targeting c-Rel can be used alone in anti-tumor therapy, and can also be used in combination with anti-PD-1 antibody therapy or CAR-T therapy to increase the immunosuppressive microenvironment in tumor tissues and enhance the anti-tumor effects of anti-PD-1 or CAR-T cells. The present invention constructs an oncolytic adenovirus carrying shRNA of the c-Rel gene based on adenovirus, and replaces the original promoter of the virus with the hTERT promoter. The telomerase activity of most normal human cells is inhibited, but high expression of hTERT and activation of telomerase can be observed in more than 85% of human malignant tumors, and it is suitable for more tumors than H101. H101 is only suitable for tumors with p53 mutations. And the shRNA sequence of the c-Rel gene is inserted downstream of the U6 promoter to obtain an oncolytic virus with oncolytic and knockdown of the c-Rel gene. Although the oncolytic adenovirus H101 can recruit immune cell infiltration into the tumor microenvironment, due to the too strong immunosuppressive effect of the tumor microenvironment, a good therapeutic effect still cannot be achieved. However, inserting shRNA with silencing c-Rel gene expression enables the virus to further infect immunosuppressive cells in the tumor microenvironment, especially myeloid-derived immunosuppressive cells and silence the expression of c-Rel during oncolysis, weakening the immunosuppressive effect of the tumor microenvironment and remodeling the anti-tumor immune response of the body, so as to achieve the purpose of more effective treatment or cure of tumors.
[0045] It should also be noted that the present invention has successfully constructed an oncolytic adenovirus with silencing c-Rel expression, and this virus can effectively silence the expression of c-Rel in both tumor cells and MDSC cells, as Figure 2 shown. In in vitro experiments, it was found that neither Ad-shNC nor Ad-shc-Rel oncolytic adenovirus had a significant effect on the viability of MDSC cells even when the MOI was 2000 and the treatment time was up to 96 hours, as Figure 3 shown, which indicates that the replication of the virus in normal cells is restricted. In in vitro experiments, both of these oncolytic adenoviruses had good lysis effects on tumor cells. However, silencing the expression of c-Rel did not further enhance their oncolytic effects, as Figure 4 shown. Adding Ad-shNC and Ad-shc-Rel oncolytic adenoviruses during the in vitro induction of MDSC cells did not affect the proportion of bone marrow-derived cells induced into MDSC cells, as Figure 5 shown. When the ratio of MDSC:T cells was 1:1 and 1:2, compared with the control virus, silencing the expression of c-Rel could more significantly reverse the immunosuppression of MDSC cells and release the proliferation of T cells, as Figure 5As shown. In in-vivo experiments, compared with the control oncolytic adenovirus Ad-shNC, the oncolytic adenovirus Ad-shc-Rel could significantly delay the growth of murine melanoma B16-F10, as shown in (A) of Figure 5 ; reduce the tumor weight, as shown in (B) of Figure 5 ; and prolong the survival time of tumor-bearing mice, as shown in (C) of Figure 5 .
[0046] Specifically, as shown in Figure 2 , the oncolytic adenovirus Ad-shc-Rel successfully silenced the expression of c-Rel in tumor cells and MDSC cells. Ad-shc-Rel could silence the expression of c-Rel in B16-F10 (A), Panc02 (B), EL-4 (C), MDSC (D) cells, and its silencing efficiency increased with the increase of MOI. As shown in Figure 3 , the oncolytic adenoviruses Ad-shNC and Ad-shc-Rel did not affect the viability of MDSC cells. The oncolytic adenoviruses Ad-shNC and Ad-shc-Rel were co-cultured with MDSC cells for 24 hours (A), 48 hours (B), 72 hours (C) and 96 hours (D) when MOI ranged from 0 - 2000, and had no effect on the viability of MDSC cells, indicating that hTERTp restricted the replication of oncolytic adenovirus in normal cells. As shown in Figure 4 , the oncolytic adenoviruses Ad-shNC and Ad-shc-Rel had the same lytic effect on tumor cells. The oncolytic adenoviruses Ad-shNC and Ad-shc-Rel were co-cultured with tumor cells B16-F10 (A - B), Panc02 (C - D), EL-4 (E - F) for 48 - 72 hours when MOI ranged from 0 - 2000, and the tumor cells showed varying degrees of lysis. With the increase of MOI, the ability of oncolytic adenovirus to lyse tumor cells increased, manifested as a decrease in cell viability, but the ability of these two viruses to lyse the same kind of tumor cells was comparable, indicating that silencing the expression of c-Rel on tumor cells did not further improve the ability of oncolytic virus to lyse tumor cells. As shown in Figure 5As shown, oncolytic adenovirus Ad-shc-Rel silencing c-Rel expression can reverse the inhibitory function of MDSC cells. During the process of inducing bone marrow-derived cells into MDSC cells, in the presence of Medium (A), oncolytic adenovirus Ad-shNC (B), and oncolytic adenovirus Ad-shc-Rel (C), there was no effect on the proportion of MDSC cells in total viable cells. (A-C) are typical representative flow cytometry plots, and (D) are statistical data. When the MDSC:T cell ratio was 1:1, compared with Ad-shNC, silencing c-Rel expression could significantly reverse the immunosuppressive property of MDSC cells and release the proliferation of T cells. (E) is a typical representative flow cytometry plot, and (F) are statistical data. When MDSC:T cells were 1:2, compared with Ad-shNC, silencing c-Rel expression could also significantly reverse the immunosuppressive property of MDSC cells and release the proliferation of T cells. (E) is a typical representative flow cytometry plot, and (F) are statistical data. As Figure 6 shown, oncolytic adenovirus Ad-shc-Rel can enhance the effect against B16-F10 murine melanoma. C57 / BL6 WT mice at 7-8 weeks old were inoculated with 5×105 B16-F10 melanoma cells. When the tumor size was approximately 100 mm3, the mice were randomly divided into 3 groups and were injected intratumorally with PBS, Ad-shNC, and Ad-shc-Rel respectively. The dosage of the oncolytic virus was 1×109 PFU, and the injection volume was 50 mL. (A) Tumor growth curve, (B) Tumor weight, (C) Survival time of tumor-bearing mice.
[0047] The above-described embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention and should all be included within the protection scope of the present invention.
Claims
1. A method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus, characterized in that, the method comprises: designing shRNA sequences of c-Rel gene, and screening out target shRNA sequences from each of the shRNA sequences according to the silencing effect of the shRNA sequences; constructing by using pDC315 plasmid, hTERT promoter, U6 promoter and target shRNA sequence to obtain a shuttle plasmid; packaging adenovirus according to the shuttle plasmid to obtain an oncolytic adenovirus carrying shRNA of c-Rel gene.
2. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to claim 1, characterized in that, the step of constructing by using pDC315 plasmid, hTERT promoter, U6 promoter and target shRNA sequence to obtain a shuttle plasmid comprises: replacing the original CMV promoter of pDC315 plasmid with hTERT promoter, and inserting the replaced pDC315 plasmid into U6 promoter to obtain pDC315-hTERT-E1A-U6 plasmid; inserting the target shRNA sequence into pDC315-hTERT-E1A-U6 plasmid to obtain a shuttle plasmid.
3. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to claim 2, characterized in that, the shuttle plasmid is pDC315-hTERTp-E1A-EGFP-U6-mRel shRNA.
4. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to claim 3, characterized in that, the step of packaging adenovirus according to the shuttle plasmid to obtain an oncolytic adenovirus carrying shRNA of c-Rel gene comprises: packaging adenovirus by using 293 cells for virus packaging and virus plasmid to obtain the oncolytic adenovirus, wherein the virus plasmid comprises the shuttle plasmid and a backbone plasmid.
5. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to claim 4, characterized in that, the backbone plasmid is pBHGlox(delta)E1,3Cre.
6. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to any one of claims 1 to 5, characterized in that, the oncolytic adenovirus is Ad5-hTERTp-EGFP-mRel shRNA.
7. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to claim 1, characterized in that, the shRNA sequence of c-Rel gene is the shRNA sequence of mammalian c-Rel gene.
8. The method for constructing an oncolytic adenovirus carrying shRNA of c-Rel gene based on adenovirus according to claim 7, characterized in that, The mammal is a mouse, and the shRNA of the c-Rel gene of the mouse is 5'-GGATTAGTGCAGGAATCAATC-3', or 5'-GGATCTGCCGTGTGAACAAGA-3', or 5'-GGATCAACTGGAGAAGGAAGA-3'.
9. The method for constructing an oncolytic adenovirus carrying shRNA of the c-Rel gene based on an adenovirus according to claim 1, characterized in that, after the step of packaging an adenovirus according to the shuttle plasmid to obtain an oncolytic adenovirus carrying shRNA of the c-Rel gene, it further includes: replacing the telomerase promoter of the oncolytic adenovirus with the promoter of E1A of human adenovirus type 5, deleting the adenovirus E3 region of the oncolytic adenovirus, and inserting the shRNA sequence of the c-Rel gene after the oncolytic adenovirus.
10. The application of the oncolytic adenovirus carrying shRNA of the c-Rel gene according to claim 1 in the preparation of a drug for treating malignant tumors.