A system for controlling viral replication in response to hypoxic environments and its applications

Abstract

This invention provides a system for controlling viral replication in response to hypoxic environments and its application. The system comprises: a gene encoding a hypoxia-responsive element, a gene encoding a viral transcription factor recognition element, and one or more genes encoding viral repressor factors, connected sequentially. This allows the viral repressor factor, a product of the target gene, to be expressed under normal oxygen conditions, but expressed at low levels or not at all under hypoxic conditions, thereby enabling the oncolytic virus to exert its specific killing effect on cancer cells and improving the efficacy of oncolytic viruses in the treatment of hypoxic diseases such as solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a system for controlling viral replication in response to hypoxic environments and its use in the preparation of drugs for treating hypoxic diseases, such as tumors. Background Technology

[0002] Oncolytic virus-mediated immunotherapy is another important emerging cancer treatment strategy following the three major conventional treatments (surgery, radiotherapy, and chemotherapy) and immunotherapy. Other cancer immunotherapies, such as cellular immunotherapy, are expensive and struggle to address the issue of universality. In contrast, oncolytic viruses offer advantages such as high killing efficiency, good targeting, fewer adverse reactions, diverse tumor-killing pathways, low drug resistance, and low cost. Studies have shown that oncolytic viruses can be applied to different types of tumors or tumors at different stages of progression, and can even improve the overall survival rate of patients with metastatic or incurable cancers. For patients with advanced cancer, oncolytic virus therapy is considered one of the most important life-saving methods, and oncolytic virus treatment can induce complete tumor regression or remission. Currently, oncolytic virus therapy is receiving widespread attention and has a very broad prospect in the treatment of cancer.

[0003] In the early 20th century, oncolytic virus therapy began to be introduced into cancer treatment strategies. To date, oncolytic viruses have evolved from the initial, unmodified products—those directly utilizing wild-type or attenuated wild-type virus strains, such as reovirus and Newcastle disease virus—to the stage of developing genetically modified virus strains. Viruses that can proliferate within tumor cells through gene recombination include herpes simplex virus, adenovirus, measles virus, and vaccinia virus. Furthermore, they have iterated and upgraded to the third generation of highly potent oncolytic viruses, namely those involving gene insertion and synergistic combination therapy. This is currently the main research direction for oncolytic viruses in clinical practice.

[0004] However, in its nearly 100-year development, oncolytic viruses have faced limitations due to their own inherent constraints, the heterogeneity of tumor tissues, and the complexity of tumor cells. Although several oncolytic virus drugs have been launched, their therapeutic effects have been minimal, and the efficacy of monotherapy has failed to meet clinical expectations. Consequently, their application has been questioned, significantly limiting their clinical use in solid tumors. Current improvement strategies involve modifying the genes or delivery methods of oncolytic viruses to adapt them to the patient's system and specifically activate the immune system for targeted tumor killing; or combining oncolytic viruses with immune checkpoint inhibitors such as PD-1 and CTLA-4, leveraging the oncolytic virus to disrupt the tumor tissue microenvironment, allowing these immune checkpoint inhibitors to exert their effects more effectively.

[0005] The major challenge facing oncolytic virus therapy lies not only in identifying highly sensitive viral strains and matching indications, but also in the virus itself. As foreign pathogens to the body, oncolytic viruses inevitably trigger an immune response, regardless of how their activity is reduced. Safe regulatory mechanisms are needed to ensure that oncolytic viruses replicate to the maximum extent within tumor cells while not replicating in normal cells, thereby improving tumor targeting specificity while maintaining safety.

[0006] The relatively hypoxic tumor microenvironment of solid tumors is an ideal target for gene therapy. Hypoxia signals can induce oncolytic viruses to replicate exclusively within tumor tissue, thereby enhancing the specificity of tumor treatment and minimizing damage to normal tissues. Studies have shown that under hypoxic conditions, the expression level of hypoxia-inducible factor-1 (HIF-1) produced in the cell nucleus increases, leading to increased binding to target genes and promoting their transcription, thus triggering a series of cellular responses to hypoxia. More than 60 target genes of HIF-1 have been identified, all of which possess hypoxia-responsive elements (HREs). HREs consist of the HIF1 binding site (a shared sequence of 5'-TACGTGCT-3') and flanking functional sequences. Mutations at the HIF1 binding site result in the loss of the gene's hypoxia-sensitive transcriptional response. Therefore, hypoxia-responsive elements can be used as control elements to initiate gene expression. Summary of the Invention

[0007] The purpose of this invention is to provide a hypoxia-sensitive transcriptional control system that enables the viral inhibitory factor, which is the product of the target gene (GOI), to be expressed in a normal oxygen environment, but expressed at a low level or not expressed in a hypoxic environment, thereby exerting the specific killing effect of oncolytic viruses on cancer cells and improving the efficacy of oncolytic viruses in the treatment of hypoxic diseases such as solid tumors.

[0008] On one hand, the present invention provides a system for controlling viral replication in response to hypoxic environments, comprising: a gene encoding a hypoxia response element (HRE), a gene encoding a virus transcription promoter (VTP), and one or more genes encoding a virus inhibitory factor (VIF) connected in sequence.

[0009] The system for controlling viral replication in response to hypoxia according to the present invention enables high expression of hypoxia-sensitive viral suppressor proteins under normal tissue conditions such as normoxic conditions, thereby inhibiting viral replication; while under hypoxic conditions, the viral suppressor proteins are expressed at low levels, thus relieving the inhibitory effect on viral replication. Viral replication can be regulated by initiating efficient transcriptional expression of target genes in conjunction with these genes. This transcriptional control system enhances viral safety without affecting its efficacy. Using the hypoxia-sensitive transcriptional control system provided by the present invention, different target genes can be introduced to treat hypoxic diseases such as solid tumors and to prepare corresponding drugs.

[0010] In this invention, the hypoxic environment refers to cells or tissues with an oxygen content of less than 2%. Tumor tissues and cells have a high demand for energy substances such as oxygen and glucose. As the blood supply to tumor tissues is insufficient, hypoxia occurs in tumor cells and tissues, and the oxygen content of their cells and tissues is often less than 2%.

[0011] According to some embodiments of the present invention, the nucleotide sequence of the gene encoding the hypoxia response element is as shown in SEQ ID NO: 1, or is a repeating series of more than one, preferably three, more preferably five, nucleotide sequences as shown in SEQ NO: 1.

[0012] According to some embodiments of the present invention, the viral transcription factor recognition element is a viral transcription promoter. Preferably, the viral transcription promoter is selected from one or more of PE / L, truncated PEL / L (PE / LL), further truncated PE / L (PE / LS), P7.5, truncated P7.5 (P7.5-71), further truncated P7.5 (P7.5-24), or LTRminiP.

[0013] According to some specific embodiments of the present invention, the nucleotide sequence of PE / L is shown in SEQ ID NO: 2; the nucleotide sequence of PE / LL is shown in SEQ ID NO: 3; the nucleotide sequence of PE / LS is shown in SEQ ID NO: 4; the nucleotide sequence of P7.5 is shown in SEQ ID NO: 5; the nucleotide sequence of P7.5-71 is shown in SEQ ID NO: 6; the nucleotide sequence of P7.5-24 is shown in SEQ ID NO: 7; and the nucleotide sequence of LTRminiP is shown in SEQ ID NO: 8.

[0014] According to some embodiments of the present invention, the viral inhibitory factor is interferon. The interferon may be selected from one or more of IFN-β, α1, α2, γ, or κ. Preferably, the interferon is IFN-β and / or IFN-γ. More preferably, the nucleotide sequence of the IFN-β is as shown in SEQ ID NO: 21, and / or its amino acid sequence is as shown in SEQ ID NO: 22. More preferably, the nucleotide sequence of the IFN-γ is as shown in SEQ ID NO: 23, and / or its amino acid sequence is as shown in SEQ ID NO: 24.

[0015] According to some preferred embodiments of the present invention, the system for controlling viral replication in response to hypoxic environments comprises two or more interconnected genes encoding viral repressor factors, and adjacent genes encoding viral repressor factors include a gene encoding a self-splicing linker strand. Preferably, the self-splicing linker strand is a 2A short peptide. Specifically, the 2A short peptide may be selected from one or more of porcine cephalovirus P2A, equine rhinitis virus E2A, foot-and-mouth disease virus F2A, or vesicular tussock moth β tetrasomal virus T2A, preferably porcine cephalovirus P2A. Preferably, the system for controlling viral replication in response to hypoxic environments comprises a gene encoding IFN-β-P2A-IFN-γ. More preferably, the nucleotide sequence of the gene encoding IFN-β-P2A-IFN-γ is as shown in SEQ ID NO: 11, and / or the amino acid sequence of IFN-β-P2A-IFN-γ is as shown in SEQ ID NO: 12.

[0016] According to some embodiments of the present invention, a gene encoding a linker is included between the gene encoding the hypoxia response element and the gene encoding the viral transcription factor recognition element, and between the gene encoding the viral transcription factor recognition element and the gene encoding the viral repressor factor. Preferably, the linker is (GGS). n , where n is 1-3.

[0017] According to some preferred embodiments of the present invention, the system for controlling viral replication in response to hypoxic environments includes genes encoding constructs such as:

[0018] 5* Hypoxia Response Elements -PE / L-IFN-β-P2A-IFN-γ. Here, "5* Hypoxia Response Elements" refers to five consecutively repeating hypoxia response elements.

[0019] On the other hand, the present invention provides a carrier comprising a system according to the invention for controlling viral replication in response to hypoxic environments.

[0020] According to some embodiments of the present invention, the vector is selected from one or more of vaccinia virus, adenovirus, herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-2), vesicular stomatitis virus, echovirus, reovirus, alphavirus, yellow fever virus, Coxsackie virus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus or retrovirus, preferably vaccinia virus.

[0021] On the other hand, the present invention provides a recombinant virus whose genome contains a system according to the invention for controlling viral replication in response to hypoxic environments.

[0022] According to some embodiments of the present invention, the genome of the recombinant virus further includes the knockout of a hypoxia-sensing gene and / or an interferon-repressing gene. Preferably, the hypoxia-sensing gene is the C16 hypoxia-sensing gene, and / or the interferon-repressing gene is the C9 interferon-repressing gene.

[0023] According to some embodiments of the present invention, the genome of the recombinant virus further includes one or more selected from co-stimulatory molecules, cytokines, negatively modulating molecules and signaling pathway blocking antibodies, chemokines or cytotoxic molecules, preferably cytotoxic molecules, and more preferably the coding gene of BiTE.

[0024] In another aspect, the present invention provides a pharmaceutical composition comprising a system according to the invention for controlling viral replication in response to hypoxic environments, a vector according to the invention or a recombinant virus according to the invention, and optionally pharmaceutically acceptable excipients.

[0025] According to some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from one or more of sodium phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, sorbitol, inositol, an aqueous solution of poloxamer 188 at a mass ratio of 0.001%, an aqueous solution of poloxamer 188 at a mass ratio of 0.005%, tris(hydroxymethyl)aminomethane (Tris), magnesium chloride, or water for injection.

[0026] In another aspect, the present invention provides the use of a system for controlling viral replication in response to hypoxic environments according to the present invention, a vector according to the present invention, a recombinant virus according to the present invention, or a pharmaceutical composition according to the present invention in the preparation of a medicament for treating hypoxic diseases, such as cancer.

[0027] According to some embodiments of the present invention, the cancer is selected from one or more of blood cancers, such as leukemia; or lymphoma.

[0028] According to some embodiments of the present invention, the cancer is a solid tumor. Preferably, the solid tumor is selected from one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal carcinoma, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, gastrointestinal stromal tumor (GIST), bone cancer, prostate cancer, thyroid carcinoma, or brain cancer.

[0029] According to some embodiments of the present invention, the drug is an oncolytic virus drug.

[0030] According to some embodiments of the present invention, the drug is an injectable preparation.

[0031] Accordingly, the present invention provides a method for treating hypoxic diseases, such as cancer, comprising administering to a subject in need a therapeutically effective amount of a system for controlling viral replication in response to a hypoxic environment according to the present invention, a vector according to the present invention, a recombinant virus according to the present invention, or a pharmaceutical composition according to the present invention.

[0032] According to some embodiments of the present invention, the cancer is selected from one or more of blood cancers, such as leukemia; or lymphoma.

[0033] According to some embodiments of the present invention, the cancer is a solid tumor. Preferably, the solid tumor is selected from one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal carcinoma, small bowel cancer, large bowel cancer, colorectal cancer, bladder cancer, gastrointestinal stromal tumor (GIST), bone cancer, prostate cancer, thyroid carcinoma, or brain cancer.

[0034] According to some embodiments of the present invention, the administration is performed by intratumoral injection and / or intravenous administration.

[0035] This invention provides a combination of molecular elements and their applications that enable viral replication under hypoxic conditions to be superior to that under normoxic conditions. The hypoxia-sensitive transcriptional control system provided by this invention consists of a hypoxia-responsive element, a viral transcription factor recognition element, and a viral inhibitory factor element. This system has the following three major advantages: 1) It expresses viral inhibitory factors under normal oxygen conditions, while under hypoxic conditions, it expresses low-level or no viral inhibitory factors, ensuring the system's hypoxia sensitivity; 2) Under normal oxygen conditions, viral transcription factors can specifically bind to their corresponding recognition elements, and viral inhibitory factors are transcribed and expressed, interfering with viral replication, thereby avoiding severe toxic side effects caused by systemic administration, and possessing the advantages of strong tumor selectivity, low toxicity, and high bioavailability; 3) Under hypoxic conditions, viral transcription factors cannot bind to their corresponding recognition elements, and viral inhibitory factors are not expressed or expressed at low levels, relieving the inhibitory effect on viral replication, allowing the virus to achieve efficient replication and proliferation; 4) The virus can also carry other exogenous target genes, which can be efficiently expressed along with viral replication, exerting their maximum activity.

[0036] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0037] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0038] Figure 1 ae shows the hypoxia-regulating effect of the HRE-VTP-GOI mode. Figure 1 a is a map showing the plasmid containing HRE-PE / L-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus; Figure 1 b is a map showing the plasmid containing HRE-PE / LL-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus; Figure 1 c is a map of the plasmid HRE-PE / LS-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus; Figure 1 Image d shows a map of the plasmid containing HRE-LTRminiP-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus. Using recombination technology, the plasmid was... Figure 1 The gene in the plasmid shown in ad was inserted into the vaccinia virus genome. Cells were infected for 24 hours under normoxic (21% O2) and hypoxic (1% O2) conditions. Compared to normoxic conditions, luciferase expression was downregulated under hypoxic conditions. Figure 1 e).

[0039] Figure 2 ae shows the hypoxia-regulating effect of the HRE-VTP-GOI mode. Figure 2 a is a map of the plasmid containing HRE-P7.5-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus; Figure 2 b is a map of the plasmid containing HRE-P7.5-71-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus; Figure 2 c is a map of the plasmid containing HRE-P7.5-24-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus; Figure 2 Image d shows a map of the plasmid containing HRE-LTRminiP-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus. Using recombination technology, the plasmid was... Figure 2 The gene of the plasmid shown in ad was inserted into the vaccinia virus genome. Cells were infected for 24 hours under normoxic (21% O2) and hypoxic (1% O2) conditions. Compared to normoxic conditions, luciferase expression was downregulated under hypoxic conditions. Figure 2 e).

[0040] Figure 3 Displayed in human thymkinase-deficient cell line TK143 - In the study, at a concentration of 3 ng / mL, IFN-β, α1, α2, γ, and κ inhibited the replication of vaccinia virus (Tiantan strain). Compared with the untreated group, IFN-β most significantly inhibited vaccinia virus infection.

[0041] Figure 4 Ac showed that knocking out the C9 gene of vaccinia virus increased sensitivity to interferon. Using recombination technology, HRE-PE / L-IFN-β-P2A-INF-γ was inserted into the vaccinia virus genome, and then the C9 gene in the vaccinia virus genome was knocked out using recombination technology. TK143 cells were then infected. - When cells were infected under normoxic conditions for 24 hours, it was observed that the vaccinia virus with the C9 gene knocked out was difficult to replicate in large quantities, and its plaque size was much smaller than that of the virus without the C9 gene knocked out.

[0042] Figure 5 Ac showed that simultaneous knockout of the hypoxia-sensing gene (C16) and the viral interferon-suppressing gene (C9), along with integration of the HRE-PE / L-IFN-β-P2A-INF-γ gene, mediates specific viral replication under hypoxic conditions. Among these, Figure 5 a is a map of the plasmid loaded with HRE-PE / L-IFN-β-P2A-INF-γ at the VGF2 site; Figure 5bc is a map of plasmids that knock out vaccinia virus C16 and C9; Figure 5 d shows that HRE-PE / L-IFN-β-P2A-INF-γ was inserted into the vaccinia virus genome using recombination technology, while C16 and C9 genes were knocked out. Cells were infected under normoxic and hypoxic conditions for 24 hours. It was observed that the virus replicated extensively under hypoxic conditions, and its plaque size was much larger than that of the virus under normoxic conditions (4 viral monoclonal strains were obtained).

[0043] Figure 6 The study demonstrated the specific killing effect of a viral strain that simultaneously knocked out the hypoxia-sensing gene (C16) and the viral interferon-suppressing gene (C9) and integrated HRE-PE / L-IFN-β-P2A-INF-γ on tumor cells under both normoxic and hypoxic conditions. Under hypoxic conditions, the killing effect of this strain was significantly higher than that under normoxic conditions.

[0044] Figure 7 The study showed that intratumoral injection of an oncolytic virus (TTV-β / γ-C9 / C16-KO) controlled by a hypoxia-sensitive transcriptional regulatory gene can effectively inhibit tumor growth. Detailed Implementation

[0045] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with these descriptions. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods in the art. Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents purchased from the sales companies. DMEM culture medium was purchased from Corning. Fetal bovine serum was purchased from BI. The LIPOFECTAMINE 3000 transfection kit was purchased from Thermo Fisher Scientific. Gene synthesis was performed by Shanghai Jierui Biotechnology Co., Ltd. Stabl3 chemocompetent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. Endotoxin-free plasmid miniprep kits were purchased from OMEGA. TK143 - Cells and SKOV3 ovarian cancer cells were purchased from ATCC (USA). A fluorescence microscope was purchased from Nikon (Japan). Luciferase substrates were purchased from Promega Biotechnology Co., Ltd. A GloMax96 microplate luminescence detector was purchased from Promega Biotechnology Co., Ltd.

[0047] Example 1: Construction of recombinant plasmid for vaccinia virus

[0048] The nucleotide sequences shown in SEQ ID NO: 13-19 were synthesized by Shanghai Jereh Biotechnology Co., Ltd., and cloned into the VFG2 gene recombinant plasmid (pSC65-VFG-2) to obtain the following recombinant plasmids: HRE-PE / L-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 13; HRE-PE / LL-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 14; HRE-PE / LS-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 15; HRE-P7.5-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 16; HRE-P7.5-71-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 17; HRE-P7.5-24-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 18; and HRE-PE / LL-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 19; and HRE-PE / LL-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 15; HRE-PE / LS-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 16; HRE-P7.5-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 17; and HRE-P7.5-24-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 18; and HRE-PE / LL-Luciferase-mCherry carrying the nucleotide sequence shown in SEQ ID NO: 19 ... The HRE-LTRminP-Luciferase-mCherry recombinant plasmid with the nucleotide sequence shown in SEQ ID NO: 19 (where the nucleotide sequence of Luciferase is shown in SEQ ID NO: 9 and its amino acid sequence is shown in SEQ ID NO: 10) was used. All of the above recombinant plasmids used red fluorescence as the recombinant screening signal, and their plasmid maps are shown below. Figure 1 ad and Figure 2 As shown in the image above.

[0049] The nucleotide sequence shown in SEQ ID NO: 20 was synthesized by Shanghai Jereh Biotechnology Co., Ltd., and cloned into the VFG2 gene recombinant plasmid (pSC65-VFG-2) to obtain the pSC65-VGF-2-HRE-PE / L-IFN-β-P2A-INF-γ-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ NO: 20. The red fluorescent signal was used as the recombinant selection signal for this recombinant plasmid, and its plasmid map is shown below. Figure 4 As shown in a.

[0050] C9 and C16 gene knockout recombinant plasmids, such as Figure 5 As shown in b and 5c, it does not carry foreign genes, and the C9 and C16 gene sites are replaced with selection markers, namely the blue fluorescent protein and green fluorescent protein genes, respectively.

[0051] Example 2: Recombination, purification, and titer determination of vaccinia virus

[0052] 1. The vaccinia virus gene recombinant plasmid constructed using Example 1 (e.g., Figure 1Recombination of vaccinia virus was performed as shown in (ad, 2a-d, 4a, 5b-c).

[0053] 1.1 Cell preparation: TK143 - Cells were seeded in 6-well plates, approximately 1 × 10⁶ cells per well. 6 After culturing for approximately 24 hours, when the cells have adhered to the wall and covered the entire bottom surface, proceed to the next step.

[0054] 1.2 Vaccine virus incubation: Cells were infected with wild-type vaccinia virus Tian Tan strain at 0.0125 / 3 PFU (PFU: plaque-forming units, virus titer) / cell. After incubation at 37°C for 1 hour, the cells were removed, the supernatant was aspirated, and the cells were washed once with 1 mL PBS. Then, 1 mL of DMEM complete medium (medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin antibiotics (PS)) was added.

[0055] 1.3 Plasmid transfection: TK143 was transfected with a vaccinia virus gene recombinant plasmid. - Cells. Incubate at 37°C for approximately 48 hours, the exact time depending on the cytopathic effect.

[0056] 1.4 Prepare 2×DMEM maintenance medium (DMEM medium + 2% FBS + 1% PS) for virus plaque preparation, and add 2% preheated low melting point agarose.

[0057] 1.5 Remove the supernatant from the 6-well plate, add 4 mL of the speckle-laying mixture to each well (300 μL). Then carefully place the plate in a 4°C freezer to promote solidification. Once the low-melting-point agarose has solidified, transfer it to a 37°C incubator for further incubation.

[0058] 1.6 Under a fluorescence microscope, pick recombinant viral plaques and add 500 μL of DMEM complete culture medium. Repeat the freeze-thaw cycle at -80°C at least three times to release as much virus as possible. This purification process needs to be performed at least five times.

[0059] 1.7 Perform small-scale amplification of recombinant vaccinia virus, using TK143. - Cells were grown in a six-well plate, 1 × 10⁶ cells per well. 6 Each cell is used when the area of ​​the well plate is approximately 100% of the bottom area.

[0060] 1.8 Before inoculating with the virus, replace the culture medium in the wells with 2 mL of DMEM maintenance medium. Repeatedly pipette the purified, fluorescently-containing virus solution until it disperses. Add approximately 100 μL of virus solution to each well. Incubate at 37°C for approximately 48 hours, and collect the samples based on the formation of viral plaques.

[0061] 1.9 Sample Collection: Carefully aspirate 1 mL of the culture medium supernatant from the well. Use the remaining 1 mL of culture medium to thoroughly blow off the cells and collect them in an EP tube. This can be used for subsequent genome extraction and as a seed for amplification.

[0062] 2. Amplification and purification of recombinant vaccinia virus

[0063] 2.1 VERO cell plating: Take 10cm dishes, approximately 5×10 cells per dish. 6 The number of cells should be such that the cell density reaches 100% when the vaccinia virus is inoculated the next day;

[0064] 2.2 Before virus inoculation, replace the DMEM complete medium with 8 mL of DMEM maintenance medium. Inoculate the cells in the DMEM maintenance medium with the virus at an inoculation volume of approximately 0.02 MOI (MOI = virus PFU / cell number). Continue culturing in an incubator at 37°C and 5% CO2 for approximately 48 hours. Collect samples based on the formation of viral plaques.

[0065] 2.3 Collecting vaccinia virus: Discard 8 mL of culture medium in the dish, take 2 mL of DMEM maintenance medium, blow off the remaining cells, and collect them in a 15 mL centrifuge tube;

[0066] 2.4 After freezing for 24 hours, the collected virus solution was repeatedly frozen and thawed twice. It was then subjected to density gradient centrifugation with 36% sucrose solution at 16000g and 4℃ for 90 min. The supernatant was carefully discarded, and the virus precipitate in the centrifuge tube was dissolved with PBS buffer. The tubes were then aliquoted and stored at -80℃ until the virus titer was determined.

[0067] 3. Titer determination of recombinant vaccinia virus

[0068] 3.1TK143 - Cell preparation: TK143 - Cells were seeded in 24-well plates, approximately 2 × 10⁶ cells per well. 5 Each cell, when used, achieves a cell density of 100% of the bottom area of ​​a 24-well plate;

[0069] 3.2 Dilute the virus. Dilute the vaccinia virus solution with DMEM maintenance medium, starting from 1:100 and performing 10-fold serial dilutions to a final volume of 1100 μL.

[0070] 3.3 Discard the DMEM complete medium in the 24-well plate, add 500 μL of diluted virus solution to each well, and perform two replicates. Incubate at 37°C and 5% CO2 for approximately 48 hours, adjusting the plaque formation time according to the viral plaque formation.

[0071] 3.4 Viral plaque counting: First, observe whether the number of viral plaques decreases by a factor of ten. Then, count the number of plaques in the replicates of the two replicates of the seed virus that have only single-digit plaques. The sum of the plaque values ​​in the two wells is then multiplied by the reciprocal of the dilution corresponding to that well to obtain the viral titer in 1 mL.

[0072] Example 3: Hypoxia Regulation Effect of HRE-VTP-GOI Mode

[0073] This embodiment uses TK143 - Vaccination virus infection experiments were conducted in cells under hypoxic conditions to test the expression of the target gene by vaccinia virus in the hypoxia-sensitive transcriptional control system integrated into the viral genome.

[0074] 1. Based on recombination technology, the plasmid containing HRE-PE / L-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus (e.g., according to Example 1) was constructed. Figure 1 As shown in a), the plasmid HRE-PE / LL-Luciferase-mCherry (as shown in a) is loaded at the vaccinia virus VGF2 gene site. Figure 1 (as shown in b) The plasmid HRE-PE / LS-Luciferase-mCherry (e.g., shown in b) is loaded into the vaccinia virus VGF2 gene locus. Figure 1 (as shown in c) and plasmids loaded with HRE-LTRminiP-Luciferase-mCherry at the vaccinia virus VGF2 gene site (e.g.) Figure 1 As shown in d), the target gene was inserted into the vaccinia virus genome. The method for preparing recombinant vaccinia virus in Example 2 was used for verification.

[0075] 2. TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates. After cell adhesion on the second day, vaccinia virus infection was performed at a multiplicity of infection (MOI) of 0.02. After infection, cells were cultured for 24 hours under hypoxic conditions (1% O2 concentration) and normoxic conditions (21% O2 concentration), respectively. After culture, cells were collected, and luciferase expression was detected.

[0076] 3. Use Microplate luminescence detectors measure the luciferase activity of cells, based on... Figure 1 As shown in e, under the control of different viral promoters, hypoxia can significantly inhibit the expression of the target gene, with downregulation of 30.6, 5.85, 4.72 and 4.45 times, respectively.

[0077] Example 4: Hypoxia Regulation Effect of HRE-VTP-GOI Mode

[0078] This embodiment uses TK143 - Vaccination virus infection experiments were conducted in cells under hypoxic conditions to test the viral expression of the target gene in the hypoxia-sensitive transcriptional control system integrated with the vaccinia virus genome.

[0079] 1. Based on recombination technology, the plasmid containing HRE-P7.5-Luciferase-mCherry loaded at the VGF2 gene locus of vaccinia virus (e.g., according to Example 1) was constructed. Figure 2 As shown in a), the plasmid HRE-P7.5-71-Luciferase-mCherry was loaded into the VGF2 gene locus of vaccinia virus (as shown in a). Figure 2 (as shown in b) The plasmid HRE-P7.5-24-Luciferase-mCherry (e.g., shown in b) is loaded into the VGF2 gene locus of vaccinia virus. Figure 2 (as shown in c) and loading HRE-LTRminiP-Luciferase-mCherry (as shown in c) at the vaccinia virus VGF2 gene site. Figure 2 The target gene was inserted into the vaccinia virus genome using the plasmid shown in example d). The method for preparing recombinant vaccinia virus was then used for verification.

[0080] 2. TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates. After cell adhesion on the second day, vaccinia virus infection was performed at a multiplicity of infection (MOI) of 0.02. After infection, cells were cultured for 24 hours under hypoxic conditions (1% O2 concentration) and normoxic conditions (21% O2 concentration), respectively. After culture, cells were collected, and luciferase expression was detected.

[0081] 3. Use Microplate luminescence detectors measure the luciferase activity of cells, based on... Figure 2 As shown in e, under the control of different viral promoters, hypoxia can significantly inhibit the expression of the target gene, with downregulation of 30.6, 4.4, 9.0 and 22.1 times, respectively.

[0082] Example 5: Comparison of the inhibitory effects of interferon IFN-β, α1, α2, γ, and κ on vaccinia virus infection.

[0083] 1.TK143 - Cell preparation: Catch TK143 cells in 24-well plates. - Cells, 1×10 per well 5 Each cell, when used, achieves a cell density of 90% of the bottom area of ​​a 24-well plate;

[0084] 2. Discard the DMEM complete medium in the 24-well plate and replace it with new DMEM complete medium. Add IFN-β, α1, α2, γ and κ at a concentration of 3 ng / mL for pre-stimulation.

[0085] 3. After 24 hours, discard the DMEM complete medium in the 24-well plate and replace it with D2 medium (DMEM complete medium containing 2% FBS). Add the same concentration of interferon and vaccinia virus dilution (MOI = 0.02) as in step 2, and incubate in an incubator.

[0086] 4. After 24 hours, observe the effects of several different IFNs on cell state, viral plaque formation, and size.

[0087] The results are as follows Figure 3 As shown, compared to the other four IFNs, IFN-β at a concentration of 3 ng / mL significantly inhibited vaccinia virus replication, reducing the number of plaques from 32 in the control group without interferon to 7. Plaque size and fluorescence intensity were also significantly reduced. Furthermore, the inhibitory effect on vaccinia virus increased with increasing IFN-β concentration, without significant cell damage. Other IFNs at the same concentration did not significantly inhibit vaccinia virus replication.

[0088] Example 6: Deletion of the C9 gene of vaccinia virus significantly increases sensitivity to interferon.

[0089] The recombinant plasmid pSC65-VGF-2-HRE-PE / L-IFN-β-P2A-INF-γ-mCherry, carrying the nucleotide sequence shown in SEQ NO: 20, constructed in Example 1, and the C9 gene knockout plasmid, as well as the method for preparing recombinant vaccinia virus in Example 2, were used for verification.

[0090] TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates. After cell adhesion the following day, cells were infected with a virus at a multiplicity of infection (MOI) of 0.02. Following virus infection, cells were cultured for 24 hours under hypoxic (1% O2) and normoxic (21% O2) conditions, respectively, and cytopathic effects were observed. Cells were then observed under a fluorescence microscope 24 hours after cytopathic effects. Figure 4 As shown in c, compared with vaccinia virus without C9 gene knockout, vaccinia virus with C9 gene knockout has significantly suppressed replication level and viral plaques significantly reduced, with nearly tenfold suppression.

[0091] Example 7: Detection of Differential Replication of Vaccinia Virus with Hypoxic Transcription System

[0092] This embodiment uses TK143- Cellular infection experiments were conducted under hypoxic conditions to test the replication of viruses with hypoxic transcription systems.

[0093] The recombinant plasmid pSC65-VGF-2-HRE-PE / L-IFN-β-P2A-INF-γ-mCherry, carrying the nucleotide sequence shown in SEQ NO: 20, constructed in Example 1, along with the C9 and C16 gene knockout plasmids and the method for preparing recombinant vaccinia virus in Example 2, was used for verification.

[0094] TK143 - Cells at 2×10 5 Cells were seeded per well into 12-well plates. After cell adhesion the following day, cells were infected with a virus at a multiplicity of infection (MOI) of 0.02. Cells were infected with a recombinant vaccinia virus integrating the HRE-PE / L-IFN-β-P2A-INF-γ gene while simultaneously knocking out the C16 and C9 genes. After infection, cells were cultured for 24 hours under hypoxic (1% O2) and normoxic (21% O2) conditions, respectively. Viral plaques were observed using a fluorescence microscope after culture. The results are shown below. Figure 5 As shown in d.

[0095] Four clones, namely strains 19-1, 19-2, 19-3, and 19-4, were screened. These clones replicated normally under hypoxic conditions (1% O2 concentration) and exhibited obvious trifluorescent plaques. However, under normoxic conditions (21% O2 concentration), viral replication was inhibited, and the size of the dual-fluorescent positive plaques was significantly smaller than that of vaccinia virus cultured under hypoxic conditions. This indicates that recombinant vaccinia virus with a hypoxic transcription system and simultaneous knockout of the C9 and C16 genes can achieve normal replication under hypoxic conditions, but replicates at low levels or not at all under normoxic conditions.

[0096] Example 8: Detection of Differential Killing of Tumor Cells by Vaccine with Hypoxic Transcription System

[0097] Tumor cell killing efficiency was assessed using a luciferase-based cytotoxicity assay. First, 1×10⁻⁶ cells were used... 4SKOV3-Luc (human ovarian cancer cells modified with the firefly luciferase gene, obtained by transducing SKOV3 ovarian cancer cells with a lentivirus carrying the firefly luciferase gene) or NCI-H292-Luc (human lung cancer cells modified with the firefly luciferase gene, obtained by transducing NCI-H292 lung cancer cells with a lentivirus carrying the firefly luciferase gene) were seeded into 96-well flat-bottom black plates, 100 μL of culture medium per well, and cultured in a 37°C, 5% CO2 cell culture incubator for 18 h. The next day, when the multiplicity of infection (MOI) was 0.02, 752-1 wild-type vaccinia virus (positive control group) and the recombinant vaccinia virus prepared in Example 6 (experimental group) were added to the wells containing target cells and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 24 h, respectively. After co-culture, the cells were used... A microplate chemiluminescence assay was used to detect the luciferase activity of target cells. The formula for calculating the cell killing rate is shown below:

[0098] Cell killing rate (%) = (Luciferase activity value of uninfected vaccinia virus group - Luciferase activity value of experimental group) / Luciferase activity value of uninfected vaccinia virus group × 100

[0099] The results are as follows Figure 6 As shown, wild-type vaccinia virus 752-1 (positive control) effectively killed SKOV3 tumor cells under both normoxic and hypoxic conditions, without selective killing characteristics. Hypoxia-sensitive vaccinia virus exhibited low tumor-killing activity under normoxic conditions, with a kill rate of only 5.35%, while under hypoxic conditions it could selectively and efficiently kill SKOV3 tumor cells (kill rate reaching 25%). The specific tumor-killing activity under hypoxic conditions was nearly 5 times that under normoxic conditions.

[0100] Example 9: In vivo antitumor activity of oncolytic viruses controlled by a hypoxia-sensitive transcriptional control system

[0101] One day prior to the event, B-NDG mice housed in sterile isolators underwent localized hair removal on their backs. Hair removal cream or an animal shaving tool could be used to expose the skin at the tumor cell inoculation site. The mouse was held firmly with the left hand, simultaneously grasping its head, neck, and back skin, turning it to the left to fully expose the shaved area on the right side of its back. The right hand was then used to disinfect the area with an alcohol swab. 1 mL of the prepared SKOV3 or NCI-H292 tumor cells were mixed by pipetting with a 1 mL insulin syringe, and then 125 μL of the cell suspension (5 × 10⁻⁶) was drawn up. 6To inject 125 μL of cell suspension into mice, insert a needle at a 30°-40° angle to the skin subcutaneously, and slowly inject the cell suspension, avoiding leakage. After injecting 125 μL of cell suspension, leave the needle in place for 2-3 seconds and then quickly withdraw it. A clearly visible small bulge will appear at the injection site. Observe the tumor formation and health status of the mice every 2-3 days after cell inoculation. After tumor formation, measure the baseline tumor volume with calipers and perform subsequent experiments.

[0102] Intratumoral infusion 5×10 3 The PFU hypoxia-sensitive regulatory system used recombinant vaccinia virus (TTV-β / γ-C9 / C16-KO) integrating the HRE-PE / L-IFN-β-P2A-INF-γ gene while simultaneously knocking out the C16 and C9 genes, along with a control wild-type vaccinia virus. Tumor size was measured every 2-3 days, with the major and minor diameters of the tumor measured using calipers. The tumor volume was calculated using the following formula:

[0103] Volume = (major axis × minor axis) 2 ) / 2.

[0104] The results are as follows Figure 7 As shown, the hypoxia-sensitive transcriptional control system-controlled vaccinia virus (TTV-β / γ-C9 / C16-KO) can effectively inhibit the growth of SKOV3 (ovarian cancer) and NCI-H292 (lung cancer). The tumor inhibition rate is as high as 100% after 66 days of viral injection. Compared with the wild-type control group of vaccinia virus, it can significantly prolong the survival time of mice. All mice in the wild-type control group of vaccinia virus died after 39 days, while mice in the TTV-β / γ-C9 / C16-KO group were still alive after 60 days.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed technical content to obtain equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention. Any modifications, equivalent changes, and modifications made to the above embodiments without departing from the content of the technical solution of the present invention and based on the technical essence of the present invention still fall within the scope of the present invention.

Claims

1. A vector comprising a system for controlling viral replication in response to a hypoxic environment; wherein the vector is vaccinia virus; wherein the system for controlling viral replication in response to a hypoxic environment comprises a gene encoding a hypoxic response element, a gene encoding a viral transcription factor recognition element, and one or more genes encoding viral suppressors, which are connected in sequence; wherein the gene encoding the hypoxic response element is a repeated series of five nucleotide sequences as shown in SEQ ID NO: 1; wherein the viral transcription factor recognition element is a viral transcription promoter selected from one or more of PE / L, truncated PE / L, re-truncated PE / L, P7.5, truncated P7.5, re-truncated P7.5, or LTRminiP; the nucleotide sequence of PE / L is shown in SEQ ID NO: 2; the nucleotide sequence of truncated PE / L is shown in SEQ ID NO: 3; the nucleotide sequence of re-truncated PE / L is shown in SEQ ID NO: 4; the nucleotide sequence of P7.5 is shown in SEQ ID NO: 5; the nucleotide sequence of truncated P7.5 is shown in SEQ ID NO: 6; the nucleotide sequence of re-truncated P7.5 is shown in SEQ ID NO: 7; and the nucleotide sequence of LTRminiP is shown in SEQ ID NO: 8; wherein the one or more genes encoding viral suppressors is a gene encoding IFN-β-P2A-IFN-γ, the nucleotide sequence of which is shown in SEQ ID NO: 11, and / or the amino acid sequence of IFN-β-P2A-IFN-γ is shown in SEQ ID NO:

12.

2. The vector of claim 1, wherein, The gene encoding the hypoxic response element and the gene encoding the viral transcription factor recognition element, and the gene encoding the viral transcription factor recognition element and the gene encoding the viral suppressor, comprise a gene encoding a linker.

3. The vector of claim 2, wherein, The linker is (GGS) n wherein n is 1-3.

4. The vector of claim 1, wherein, The system for controlling viral replication in response to a hypoxic environment comprises a gene encoding a construct as follows: 5 Hypoxia response element - PE / L-IFN-beta-P2A-IFN-gamma. 5.A recombinant virus, the genome of which comprises a system for controlling viral replication in response to a hypoxic environment; wherein the recombinant virus is recombinant vaccinia virus; wherein the system for controlling viral replication in response to a hypoxic environment comprises a gene encoding a hypoxic response element, a gene encoding a viral transcription factor recognition element, and one or more genes encoding viral suppressors, which are connected in sequence; wherein the gene encoding the hypoxic response element is a repeated series of five nucleotide sequences as shown in SEQ ID NO: 1; The viral transcription factor recognition element is a viral transcription promoter selected from one or more of PE / L, truncated PE / L, re-truncated PE / L, P7.5, truncated P7.5, re-truncated P7.5, or LTRminiP; the nucleotide sequence of the PE / L is shown in SEQ ID NO: 2; the nucleotide sequence of the truncated PE / L is shown in SEQ ID NO: 3; the nucleotide sequence of the re-truncated PE / L is shown in SEQ ID NO: 4; the nucleotide sequence of the P7.5 is shown in SEQ ID NO: 5; the nucleotide sequence of the truncated P7.5 is shown in SEQ ID NO: 6; the nucleotide sequence of the re-truncated P7.5 is shown in SEQ ID NO: 7; and the nucleotide sequence of the LTRminiP is shown in SEQ ID NO: 8; The one or more genes encoding viral suppressor factors is a gene encoding IFN-β-P2A-IFN-γ, the nucleotide sequence of the gene encoding IFN-β-P2A-IFN-γ is shown in SEQ ID NO: 11, and / or the amino acid sequence of IFN-β-P2A-IFN-γ is shown in SEQ ID NO:

12.

6. The recombinant virus of claim 5, wherein, The gene encoding the hypoxia response element and the gene encoding the viral transcription factor recognition element, and the gene encoding the viral transcription factor recognition element and the gene encoding the viral suppressor factor, comprise a gene encoding a linker.

7. The recombinant virus of claim 6, wherein, The linker is (GGS) n wherein n is 1-3.

8. The recombinant virus of claim 5, wherein, The system for controlling viral replication in response to a hypoxic environment comprises a gene encoding a construct as follows: 5 Hypoxia response element - PE / L-IFN-beta-P2A-IFN-gamma.

9. The recombinant virus of claim 5, wherein, The genome of the recombinant virus further knocks out a hypoxia sensing gene and / or an interferon suppressor gene.

10. The recombinant virus of claim 9, wherein, The hypoxia sensing gene is a C16 hypoxia sensing gene, and / or the interferon suppressor gene is a C9 interferon suppressor gene.

11. The recombinant virus of claim 5, wherein, The genome of the recombinant virus further comprises a gene encoding one or more of a co-stimulatory molecule, a cytokine, a negative regulator molecule, a blocking antibody of a signaling pathway, a chemokine, or a killer molecule.

12. The recombinant virus of claim 5, wherein, The genome of the recombinant virus further comprises a gene encoding a killer molecule.

13. The recombinant virus of claim 5, wherein, The genome of the recombinant virus further comprises a gene encoding a BiTE.

14. A pharmaceutical composition comprising the vector of any one of claims 1 to 4 or the recombinant virus of any one of claims 5 to 13, and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition of claim 14, wherein, The pharmaceutically acceptable excipient is selected from one or more of sodium phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, sorbitol, inositol, a 0.001% by mass aqueous solution of poloxamer 188, a 0.005% by mass aqueous solution of poloxamer 188, tris-hydroxymethyl aminomethane, magnesium chloride, or water for injection.

16. Use of the vector of any one of claims 1 to 4, the recombinant virus of any one of claims 5 to 13, or the pharmaceutical composition of claim 14 or 15 in the manufacture of a medicament for treating a hypoxic disease. wherein The hypoxic disease is a solid tumor.

17. The use according to claim 16, wherein, The solid tumor is selected from one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, gastrointestinal stromal tumor, bone cancer, prostate cancer, thyroid cancer sarcoma, or brain cancer.

18. The use of claim 16, wherein, The drug is an injection.

Images