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WO2024082263A1 - Système de régulation de la réplication virale en réponse à un environnement hypoxique et son utilisation - Google Patents

Système de régulation de la réplication virale en réponse à un environnement hypoxique et son utilisation Download PDF

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WO2024082263A1
WO2024082263A1 PCT/CN2022/126657 CN2022126657W WO2024082263A1 WO 2024082263 A1 WO2024082263 A1 WO 2024082263A1 CN 2022126657 W CN2022126657 W CN 2022126657W WO 2024082263 A1 WO2024082263 A1 WO 2024082263A1
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virus
cancer
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gene
ifn
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徐建青
张晓燕
丁相卿
廖启彬
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Shanghai Sinobay Biotechnology Co Ltd
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/863—Poxviral vectors, e.g. entomopoxvirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Definitions

  • the present invention belongs to the field of biopharmaceuticals, and in particular relates to a system for controlling virus replication in response to a hypoxic environment and its use in preparing drugs for treating hypoxic diseases, such as tumors.
  • Oncolytic virus-mediated immunotherapy is another important emerging tumor treatment strategy after the three major conventional treatment methods (surgery, radiotherapy and chemotherapy) and immunotherapy.
  • Other tumor immunotherapies, such as cell immunotherapy are expensive and it is difficult to solve the problem of the universality of cell immunotherapy.
  • oncolytic viruses have the advantages of high killing efficiency, good targeting, small adverse reactions, diverse tumor killing pathways, low drug resistance and low cost.
  • oncolytic virus therapy is considered to be one of the important means of saving lives, and oncolytic virus therapy can induce complete regression or remission of tumors.
  • oncolytic virus therapy is receiving widespread attention and has a very broad prospect in the treatment of tumors.
  • oncolytic virus therapy began to be introduced into cancer treatment strategies.
  • oncolytic viruses have evolved from the initial generation of products that were not genetically modified, that is, directly using wild virus strains or attenuated wild virus strains, such as reovirus and Newcastle disease virus, to the research and development stage of genetically modified virus strains.
  • the viruses that can proliferate in tumor cells through genetic recombination mainly include herpes simplex virus, adenovirus, measles virus, and vaccinia virus, and have been iteratively upgraded to the third generation of new and powerful oncolytic viruses, that is, the stage of gene insertion and combined therapy enhancement, which is also the main research direction of oncolytic viruses in the current clinical stage.
  • the current improved strategy is to modify the oncolytic virus gene or its delivery method to adapt it to the patient's body system and specifically activate the immune system to achieve targeted killing of tumors; or to use oncolytic viruses in combination with immune checkpoint inhibitors such as PD-1 and CTLA-4, using oncolytic viruses to break the tumor tissue microenvironment, so that immune checkpoint inhibitors such as PD-1 and CTLA-4 can play a better role.
  • oncolytic virus therapy is not only how to find highly sensitive virus types and matching indications, but another challenge comes from the virus itself.
  • oncolytic viruses will inevitably trigger the body's immune response no matter how the viral activity is reduced.
  • Some safe regulatory mechanisms are needed to ensure that oncolytic viruses replicate in tumor cells to the greatest extent possible and do not replicate in normal cells. While improving tumor targeting specificity, their safety must also be guaranteed.
  • hypoxia signals can be used to allow oncolytic viruses to replicate only in tumor tissues to kill tumors, thereby improving the specificity of tumor therapy and avoiding or reducing damage to normal tissues.
  • hypoxia-inducible factor-1 HIF-1
  • HIF-1 hypoxia-inducible factor-1
  • target genes of HIF-1 More than 60 target genes of HIF-1 have been discovered, and they all have hypoxia responsive elements (HRE).
  • HRE consists of the HIF1 binding site (the consensus sequence is 5'-TACGTGCT-3') and functional sequences on both sides. Mutations in the HIF1 binding site result in the loss of the gene's transcriptional response to hypoxia. Therefore, the hypoxia response element can be used as a control element to initiate expression.
  • the object of the present invention is to provide a hypoxia-sensitive transcription control system, which allows the viral inhibitory factor, which is the product of the target gene (Gene of Interest, GOI), to be expressed under a normal oxygen environment, but to be lowly expressed or not expressed under 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.
  • the viral inhibitory factor which is the product of the target gene (Gene of Interest, GOI)
  • the present invention provides a system for controlling virus replication in response to a hypoxic environment, comprising: a gene encoding a hypoxia response element (HRE), a gene encoding a virus transcription factor recognition element (VTP), and one or more genes encoding a virus inhibitory factor (VIF) connected in sequence.
  • HRE hypoxia response element
  • VTP virus transcription factor recognition element
  • VIP virus inhibitory factor
  • the system for controlling virus replication in response to hypoxic environment can realize high expression of hypoxia-sensitive virus inhibitory proteins in normal tissue environments such as normoxic conditions, thereby inhibiting virus replication; and low expression of virus inhibitory proteins in hypoxic environments, thereby relieving the inhibitory effect on virus replication.
  • the replication of the virus can be regulated by linking and starting the efficient transcriptional expression of the target gene. The safety of the virus is enhanced by this transcription control system, but its effectiveness is not affected.
  • the treatment of hypoxic diseases such as solid tumors can be achieved by accessing different target genes and preparing corresponding drugs.
  • the hypoxic environment refers to cells or tissues with an oxygen content of less than 2%.
  • Tumor tissues and cells have a great demand for energy substances such as oxygen and glucose.
  • hypoxia of tumor cells and tissues occurs, and the oxygen content of cells and tissues is often less than 2%.
  • the nucleotide sequence of the gene encoding the hypoxia response element is as shown in SEQ ID NO: 1, or is a repeated series of more than one, preferably 3, and more preferably 5 nucleotide sequences as shown in SEQ NO: 1.
  • the viral transcription factor recognition element is a viral transcription promoter.
  • the viral transcription promoter is selected from one or more of PE/L, truncated PEL/L (PE/L-L), further truncated PE/L (PE/L-S), P7.5, truncated P7.5 (P7.5-71), further truncated P7.5 (P7.5-24) or LTRminiP.
  • the nucleotide sequence of the PE/L is as shown in SEQ ID NO: 2; the nucleotide sequence of the PE/L-L is as shown in SEQ ID NO: 3; the nucleotide sequence of the PE/L-S is as shown in SEQ ID NO: 4; the nucleotide sequence of the P7.5 is as shown in SEQ ID NO: 5; the nucleotide sequence of the P7.5-71 is as shown in SEQ ID NO: 6; the nucleotide sequence of the P7.5-24 is as shown in SEQ ID NO: 7; the nucleotide sequence of the LTRminiP is as shown in SEQ ID NO: 8.
  • the virus inhibitory factor is interferon.
  • the interferon can be selected from one or more of IFN- ⁇ , ⁇ 1, ⁇ 2, ⁇ or ⁇ .
  • the interferon is IFN- ⁇ and/or IFN- ⁇ .
  • the nucleotide sequence of the IFN- ⁇ is as shown in SEQ ID NO: 21, and/or its amino acid sequence is as shown in SEQ ID NO: 22.
  • the nucleotide sequence of the IFN- ⁇ is as shown in SEQ ID NO: 23, and/or its amino acid sequence is as shown in SEQ ID NO: 24.
  • the system for controlling virus replication in response to a hypoxic environment comprises two or more interconnected genes encoding virus inhibitory factors, and a gene encoding a self-cleaving connecting chain is contained between adjacent genes encoding virus inhibitory factors.
  • the self-cleaving connecting chain is a 2A short peptide.
  • the 2A short peptide can be selected from one or more of porcine teschovirus P2A, equine rhinitis virus E2A, foot-and-mouth disease virus F2A or beta-tetrasomal virus T2A of the sphenobarbital moth, preferably porcine teschovirus P2A.
  • the system for controlling virus replication in response to a hypoxic environment comprises a gene encoding IFN- ⁇ -P2A-IFN- ⁇ . More preferably, the nucleotide sequence of the gene encoding IFN- ⁇ -P2A-IFN- ⁇ is as shown in SEQ ID NO: 11, and/or the amino acid sequence of IFN- ⁇ -P2A-IFN- ⁇ is as shown in SEQ ID NO: 12.
  • 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 inhibitory factor.
  • the linker is (GGS) n , wherein n is 1-3.
  • the system for controlling viral replication in response to a hypoxic environment comprises a gene encoding the following construct:
  • 5*Hypoxia Response Element refers to 5 consecutively repeated HFEs.
  • the present invention provides a vector comprising the system for controlling virus replication in response to a hypoxic environment according to 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, coxsackievirus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus or retrovirus, preferably vaccinia virus.
  • HSV-1 herpes simplex virus type I
  • HSV-2 herpes simplex virus type II
  • vesicular stomatitis virus echovirus
  • reovirus alphavirus
  • yellow fever virus coxsackievirus
  • Newcastle disease virus measles virus
  • poliovirus Zika virus
  • lymphocytic choriomeningitis virus M1 virus
  • Marburg virus Marburg virus
  • the present invention provides a recombinant virus, the genome of which comprises the system for controlling virus replication in response to a hypoxic environment according to the present invention.
  • the genome of the recombinant virus further knocks out a hypoxia sensing gene and/or an interferon inhibitory gene.
  • the hypoxia sensing gene is a C16 hypoxia sensing gene
  • the interferon inhibitory gene is a C9 interferon inhibitory gene.
  • the genome of the recombinant virus further comprises one or more selected from costimulatory molecules, cytokines, negative regulatory molecules and blocking antibodies, chemokines or killer molecules of signal pathways, preferably killer molecules, and more preferably BiTE encoding genes.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the system for controlling viral replication in response to a hypoxic environment according to the present invention, the vector according to the present invention or the recombinant virus according to the present invention, and optionally a pharmaceutically acceptable excipient.
  • 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 (Tris), magnesium chloride or water for injection.
  • the present invention provides use of the system for controlling viral replication in response to a hypoxic environment according to the present invention, the vector according to the present invention, the recombinant virus according to the present invention or the pharmaceutical composition according to the present invention in the preparation of a drug for treating hypoxic diseases, such as cancer.
  • the cancer is selected from one or more of blood cancers, such as leukemia, or lymphoma.
  • the cancer is a solid tumor.
  • 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 (GIST), bone cancer, prostate cancer, thyroid carcinosarcoma or brain cancer.
  • GIST gastrointestinal stromal tumor
  • the drug is an oncolytic virus drug.
  • the drug is an injection.
  • the present invention provides a method for treating hypoxic diseases, such as cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the system for controlling viral replication in response to a hypoxic environment according to the present invention, the vector according to the present invention, the recombinant virus according to the present invention, or the pharmaceutical composition according to the present invention.
  • the cancer is selected from one or more of blood cancers, such as leukemia, or lymphoma.
  • the cancer is a solid tumor.
  • 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 (GIST), bone cancer, prostate cancer, thyroid carcinosarcoma or brain cancer.
  • GIST gastrointestinal stromal tumor
  • the administering is by intratumoral injection and/or intravenous administration.
  • the present invention provides a combination of molecular elements and its application that can make the replication of viruses under hypoxic conditions better than under normoxic conditions.
  • the hypoxia-sensitive transcription control system provided by the present invention is composed of a hypoxia response element, a viral transcription factor recognition element and a viral inhibitory factor element.
  • the system has the following three advantages: 1) It expresses the viral inhibitory factor under a normal oxygen environment, and it low-expresses or does not express the viral inhibitory factor under a hypoxic environment, thereby ensuring the hypoxia sensitivity of the system; 2) Under a normal oxygen environment, the viral transcription factor can specifically bind to its corresponding recognition element, and the viral inhibitory factor is transcribed and expressed, interfering with viral replication, thereby avoiding serious toxic side effects caused by systemic administration, and has the advantages of strong tumor selectivity, low toxicity and high bioavailability; 3) Under a hypoxic environment, the viral transcription factor cannot bind to the corresponding recognition element, and the viral inhibitory factor is not expressed or low-expressed, which relieves the inhibitory effect on viral replication, and the virus can achieve efficient replication and proliferation; 4) The virus can also carry other exogenous target genes, which can be efficiently expressed following viral replication to exert its maximum activity.
  • Figures 1a-e show the hypoxic regulatory effect of the HRE-VTP-GOI model.
  • Figure 1a is a map of a plasmid loaded with HRE-PE/L-Luciferase-mCherry at the VGF2 gene site of vaccinia virus;
  • Figure 1b is a map of a plasmid loaded with HRE-PE/LL-Luciferase-mCherry at the VGF2 gene site of vaccinia virus;
  • Figure 1c is a map of a plasmid loaded with HRE-PE/LS-Luciferase-mCherry at the VGF2 gene site of vaccinia virus;
  • Figure 1d is a map of a plasmid loaded with HRE-LTRminiP-Luciferase-mCherry at the VGF2 gene site of vaccinia virus.
  • Figure 2a-e shows the hypoxic regulation of the HRE-VTP-GOI model.
  • Figure 2a is a map of a plasmid loaded with HRE-P7.5-Luciferase-mCherry at the VGF2 gene site of vaccinia virus;
  • Figure 2b is a map of a plasmid loaded with HRE-P7.5-71-Luciferase-mCherry at the VGF2 gene site of vaccinia virus;
  • Figure 2c is a map of a plasmid loaded with HRE-P7.5-24-Luciferase-mCherry at the VGF2 gene site of vaccinia virus;
  • Figure 2d is a map of a plasmid loaded with HRE-LTRminiP-Luciferase-mCherry at the VGF2 gene site of vaccinia virus.
  • Figure 3 shows the inhibitory effects of IFN- ⁇ , ⁇ 1, ⁇ 2, ⁇ and ⁇ on vaccinia virus (Tiantan strain) replication in the human thymidine kinase-deficient cell line TK143-1 at a concentration of 3 ng/mL. Compared with the untreated group, IFN- ⁇ can most significantly inhibit vaccinia virus infection.
  • Figure 4a-c shows that knocking out the C9 gene of vaccinia virus can increase the sensitivity to interferon.
  • HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ was inserted into the vaccinia virus genome using recombinant technology, and then the C9 gene in the vaccinia virus genome was knocked out using recombinant technology.
  • TK143- cells were infected separately, and the cells were infected under normoxic conditions for 24 hours. It can be observed that the vaccinia virus with the C9 gene knocked out is difficult to replicate in large quantities, and its plaque size is much smaller than the plaque size of the virus without the C9 gene knocked out.
  • Figure 5a-c shows that simultaneous knockout of the hypoxia sensing gene (C16) and the viral interferon inhibitory gene (C9), and integration of the HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ gene can mediate specific replication of the virus under hypoxic conditions.
  • Figure 5a is a map of the plasmid loaded with HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ at the VGF2 site;
  • Figure 5b-c is a map of the plasmid knocking out vaccinia virus C16 and C9;
  • Figure 5d shows that HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ was inserted into the vaccinia virus genome using recombinant technology, and the C16 and C9 genes were knocked out at the same time.
  • Cells were infected under normoxic and hypoxic conditions for 24 hours. It can be observed that the virus can replicate in large quantities under hypoxic conditions, and its plaque size is much larger than the plaque size of the virus under normoxic conditions (4 virus monoclonal strains were obtained).
  • Figure 6 shows the specific killing effect of the virus strain with both hypoxia sensing gene (C16) and viral interferon inhibitory gene (C9) knocked out and HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ integrated on tumor cells under normoxic and hypoxic conditions. Under hypoxic conditions, the killing effect of the strain is significantly higher than that under normoxic conditions.
  • FIG. 7 shows that intratumoral injection of hypoxia-sensitive transcriptional regulatory gene-controlled oncolytic virus (TTV- ⁇ / ⁇ -C9/C16-KO) can effectively inhibit tumor growth.
  • TTV- ⁇ / ⁇ -C9/C16-KO hypoxia-sensitive transcriptional regulatory gene-controlled oncolytic virus
  • the experimental methods in the following examples, unless otherwise specified, are all conventional experimental methods in the art.
  • the experimental materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents purchased from sales companies.
  • DMEM culture medium was purchased from Corning.
  • Fetal bovine serum was purchased from BI.
  • LIPOFECTAMINE 3000 transfection kit was purchased from Thermo Fisher Scientific.
  • Gene synthesis was completed by Shanghai Jierui Bioengineering Co., Ltd.
  • Stabl3 chemical competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.
  • Endotoxin-free plasmid miniprep kit was purchased from OMEGA.
  • TK143 - cells and SKOV3 ovarian cancer cells were purchased from ATCC, USA.
  • Fluorescence microscope was purchased from Nikon, Japan.
  • Luciferase substrate was purchased from Promega Biotechnology Co., Ltd.
  • GloMax96 microplate luminescence detector was purchased from Promega Biotechnology Co., Ltd.
  • the nucleotide sequences shown in SEQ ID NO: 13-19 were synthesized by Shanghai Jierui Bioengineering Co., Ltd. and cloned into the VFG2 gene recombinant plasmid (pSC65-VFG-2) respectively to obtain the HRE-PE/L-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 13; the HRE-PE/L-L-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 14; the HRE-PE/L-S-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 15; and the HRE-PE/L-S-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 16.
  • HRE-P7.5-Luciferase-mCherry recombinant plasmid HRE-P7.5-71-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 17; HRE-P7.5-24-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 18; and HRE-LTRminP-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 19 (wherein the nucleotide sequence of Luciferase is shown in SEQ ID NO: 9, and its amino acid sequence is shown in SEQ ID NO: 10).
  • the above recombinant plasmids all use red fluorescent signals as recombination screening signals, and their plasmid maps are shown in Figures 1a-d and 2a-d.
  • the nucleotide sequence shown in SEQ ID NO: 20 was synthesized by Shanghai Jierui Bioengineering Co., Ltd. and cloned into the VFG2 gene recombinant plasmid (pSC65-VFG-2) to obtain the pSC65-VGF-2-HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ -mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ NO: 20.
  • the recombinant plasmid used a red fluorescent signal as a recombination screening signal, and its plasmid map is shown in Figure 4a.
  • the C9 and C16 gene knockout recombinant plasmids are shown in Figures 5b and 5c. They do not carry exogenous genes, and the C9 and C16 gene sites are replaced with screening markers, namely blue fluorescent protein and green fluorescent protein genes, respectively.
  • TK143 - cells were plated in a 6-well plate, with about 1 ⁇ 106 cells per well. After culturing for about 24 hours, when the cells adhered to the wall and covered the entire bottom surface, the next step was performed.
  • Vaccinia virus incubation Infect cells with the wild-type vaccinia virus Tiantan strain at 0.0125/3 PFU (PFU: plaque forming unit, virus titer)/cell, incubate in a 37°C incubator for 1 hour, remove the cells, aspirate the supernatant, rinse with 1 mL PBS, and then add 1 mL DMEM complete culture medium (culture medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin antibiotics (PS)).
  • PFU plaque forming unit, virus titer
  • Plasmid transfection Transfect TK143- cells with vaccinia gene recombinant plasmid and culture in a 37°C incubator for about 48 hours, the specific time depends on the cell pathological condition.
  • TK143- cells Perform small sample amplification of the recombinant vaccinia virus by plating TK143- cells in a six-well plate, with 1 ⁇ 10 6 cells per well. When used, the cells should occupy approximately 100% of the bottom area of the well plate.
  • VERO cell plating Take a 10 cm dish, with about 5 ⁇ 10 6 cells in each dish, and ensure that the cell density reaches 100% when inoculating vaccinia virus the next day;
  • MOI virus PFU/cell number
  • Collect vaccinia virus discard 8 mL of culture medium in the dish, take 2 mL of DMEM maintenance medium to blow off the remaining cells, and collect them in a 15 mL centrifuge tube;
  • TK143 cells were plated in a 24-well plate, with approximately 2 ⁇ 10 5 cells per well. The cell density should reach 100% of the bottom area of the 24-well plate when used;
  • DMEM dilute the virus solution, starting from 1:100, and make 10-fold dilutions to a final volume of 1100 ⁇ L.
  • Virus plaque counting First, observe whether the number of virus plaques decreases tenfold, then count the number of plaques in the two duplicate wells with only single-digit plaques in the inoculated virus, and get the sum of the plaque values in the two wells. Multiply it by the reciprocal value of the dilution corresponding to the well to get the titer of the virus in 1 mL.
  • a vaccinia virus infection experiment under hypoxic conditions was conducted in TK143- cells to test the expression of target genes by vaccinia viruses in which a hypoxia-sensitive transcription control system was integrated into the viral genome.
  • the plasmids constructed in Example 1 with HRE-PE/L-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus (as shown in FIG. 1a )
  • the plasmids with HRE-PE/L-L-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus (as shown in FIG. 1b )
  • the plasmids with HRE-PE/L-S-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus as shown in FIG.
  • Example 2 The method for preparing recombinant vaccinia virus in Example 2 was used for verification.
  • TK143 - cells were plated into 12-well plates at 2 ⁇ 10 5 cells/well. After the cells adhered to the wall the next day, vaccinia virus infection was performed at a virus infection multiplicity (MOI) of 0.02. After infection, the cells were cultured under hypoxic conditions (1% O 2 concentration) and normoxic conditions (21% O 2 concentration) for 24 hours. After the culture was completed, the cells were collected and the expression of luciferase was detected.
  • MOI virus infection multiplicity
  • luciferase activity value of the cells was detected by a microplate luminescence detector. As shown in Figure 1e, under the control of different viral promoters, hypoxia conditions can significantly inhibit the expression of the target gene, with a downregulation of 30.6, 5.85, 4.72 and 4.45 times, respectively.
  • a vaccinia virus infection experiment under hypoxic conditions was conducted in TK143- cells to test the expression of target genes by viruses in which the hypoxia-sensitive transcription control system is integrated into the vaccinia virus genome.
  • the plasmids constructed in Example 1 with HRE-P7.5-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus (as shown in FIG. 2a ), HRE-P7.5-71-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus (as shown in FIG. 2b ), HRE-P7.5-24-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus (as shown in FIG. 2c ), and HRE-LTRminiP-Luciferase-mCherry loaded at the VGF2 gene site of vaccinia virus (as shown in FIG. 2d ) were used to insert the target gene into the vaccinia virus genome.
  • the method for preparing recombinant vaccinia virus in Example 2 was used for verification.
  • TK143 - cells were plated into 12-well plates at 2 ⁇ 10 5 cells/well. After the cells adhered to the wall the next day, vaccinia virus infection was performed at a virus infection multiplicity (MOI) of 0.02. After infection, the cells were cultured under hypoxic conditions (1% O 2 concentration) and normoxic conditions (21% O 2 concentration) for 24 hours. After the culture was completed, the cells were collected and the expression of luciferase was detected.
  • MOI virus infection multiplicity
  • luciferase activity value of the cells was detected by a microplate luminescence detector. As shown in Figure 2e, under the control of different viral promoters, hypoxia conditions can significantly inhibit the expression of the target gene, with a downregulation of 30.6, 4.4, 9.0 and 22.1 times, respectively.
  • Example 5 Comparison of the inhibitory effects of interferons IFN- ⁇ , ⁇ 1, ⁇ 2, ⁇ and ⁇ on vaccinia virus infection
  • TK143 cells were plated in a 24-well plate at 1 ⁇ 10 5 cells per well. The cell density should reach 90% of the bottom area of the 24-well plate when used.
  • IFN- ⁇ at a concentration of 3 ng/mL had a significant inhibitory effect on the replication of vaccinia virus.
  • the number of plaques decreased from 32 plaques in the control group without interferon to 7, and the plaque size and fluorescence brightness also decreased significantly.
  • concentration of IFN- ⁇ increased, its inhibitory effect on vaccinia virus increased, and there was no obvious damage to cells.
  • Other IFNs had no significant inhibitory effect on vaccinia virus replication at the same concentration.
  • Example 6 Deletion of the C9 gene of vaccinia virus can significantly increase sensitivity to interferon
  • TK143 - cells were plated into 12-well plates at 2 ⁇ 10 5 cells/well, and the cells were infected with viruses at a virus infection multiplicity (MOI) of 0.02 after the cells adhered to the wall the next day. After the cells were infected with the virus, the cells were cultured under hypoxic conditions (1% O 2 concentration) and normoxic conditions (21% O 2 concentration) for 24 hours, and the cell lesions were observed. The cells were observed under a fluorescence microscope 24 hours after the cell lesions.
  • MOI virus infection multiplicity
  • a vaccinia virus infection experiment under hypoxic conditions was performed in TK143- cells to test the replication of viruses with hypoxic transcription systems.
  • TK143 - cells were plated in 12-well plates at 2 ⁇ 10 5 cells/well, and the cells were attached to the wall the next day and then infected with a virus infection multiplicity (MOI) of 0.02.
  • MOI virus infection multiplicity
  • the cells were infected with a recombinant vaccinia virus that integrated the HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ gene and knocked out the C16 and C9 genes.
  • the cells were cultured under hypoxic conditions (1% O 2 concentration) and normoxic conditions (21% O 2 concentration) for 24 hours, and the virus plaques were observed under a fluorescence microscope after the culture ended. The results are shown in Figure 5d.
  • the tumor cell killing efficiency was evaluated by luciferase-based cytotoxicity assay.
  • 1 ⁇ 10 4 SKOV3-Luc human ovarian cancer cells modified by firefly luciferase gene, obtained by transducing SKOV3 ovarian cancer cells with lentivirus carrying firefly luciferase gene
  • NCI-H292-Luc human lung cancer cells modified by firefly luciferase gene, obtained by transducing NCI-H292 lung cancer cells with lentivirus carrying firefly luciferase gene
  • SKOV3-Luc human ovarian cancer cells modified by firefly luciferase gene, obtained by transducing SKOV3 ovarian cancer cells with lentivirus carrying firefly luciferase gene
  • NCI-H292-Luc human lung cancer cells modified by firefly luciferase gene, obtained by transducing NCI-H292 lung cancer cells with lentivirus carrying firefly luciferase gene
  • Example 6 On the second day, when the virus infection multiplicity (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 conditions (21% O 2 ) or hypoxic conditions (1% O 2 ) for 24 h.
  • the luciferase activity of target cells was detected by a microplate luminescence detector.
  • the calculation formula for cell killing rate is as follows:
  • Cell killing rate (%) (luciferase activity value of the group not infected with vaccinia virus - luciferase activity value of the experimental group) / luciferase activity value of the group not infected with vaccinia virus ⁇ 100
  • the results are shown in Figure 6.
  • the 752-1 wild-type vaccinia virus (positive control) can effectively kill SKOV3 tumor cells under normoxic or hypoxic conditions, and has no selective killing characteristics.
  • the hypoxia-sensitive vaccinia virus has a low tumor killing activity under normoxic conditions, with a killing rate of only 5.35%, while it can selectively and efficiently kill SKOV3 tumor cells under hypoxia (killing rate of 25%).
  • the specific killing activity of tumor cells under hypoxia is nearly 5 times that under normoxic conditions.
  • Example 9 In vivo anti-tumor effect of oncolytic virus controlled by hypoxia-sensitive transcription control system
  • the B-NDG mice raised in sterile isolators were partially depilated on the back.
  • Depilatory cream or animal shaver can be used to expose the skin at the site of tumor cell inoculation.
  • the mouse was fixed with the left hand, that is, the left hand grasped the head, neck and back skin of the mouse at the same time, so that the back was turned to the left, and the shaved area on the right side of the back was fully exposed. Then, the right hand disinfected it with an alcohol cotton ball.
  • 125 ⁇ L of cell suspension (5 ⁇ 10 6 tumor cells) was drawn, and the needle was inserted into the subcutaneous part of the mouse at an angle of 30°-40° with the needle tip and the skin.
  • the cell suspension was slowly injected to avoid cell leakage.
  • the needle was quickly pulled out after 2-3 seconds. A clearly visible small bag was seen under the skin at the injection site. The tumor formation and health status of the mice were observed every 2-3 days after cell inoculation. After the tumor was formed, the baseline tumor volume was measured with a vernier caliper and subsequent experiments were performed.
  • vaccinia virus of hypoxia-sensitive regulatory system i.e., recombinant vaccinia virus integrating HRE-PE/L-IFN- ⁇ -P2A-INF- ⁇ gene and knocking out C16 and C9 genes (TTV- ⁇ / ⁇ -C9/C16-KO) and control wild-type vaccinia virus were reinfused into the tumor.
  • the tumor size was measured every 2-3 days, and the long and short diameters of the tumor were measured with a vernier caliper.
  • the tumor volume was calculated as follows:
  • the results are shown in Figure 7.
  • the vaccinia virus controlled by the hypoxia-sensitive transcriptional control system (TTV- ⁇ / ⁇ -C9/C16-KO) can effectively inhibit the growth of SKOV3 (ovarian cancer) and NCI-H292 (lung cancer).
  • the tumor inhibition rate after 66 days of virus injection is as high as 100%.
  • the survival of mice can be significantly prolonged. All the vaccinia virus mice in the wild-type control group died on the 39th day, while the mice in the TTV- ⁇ / ⁇ -C9/C16-KO group were still alive at 60 days.

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Abstract

L'invention concerne un système de régulation de la réplication virale en réponse à un environnement hypoxique et son utilisation. Le système comprend : un gène codant pour un élément de réponse à l'hypoxie, un gène codant pour un élément de reconnaissance d'un facteur de transcription viral, et un ou plusieurs gènes codant pour un facteur d'inhibition virale, lesdits gènes étant reliés de manière successive. Le système permet au facteur d'inhibition viral, qui est un produit d'un gène d'intérêt, d'être exprimé dans un environnement à oxygène normal, et d'être faiblement exprimé ou non exprimé dans un environnement hypoxique, de sorte que l'effet de destruction spécifique des virus oncolytiques sur les cellules cancéreuses est exercé, améliorant ainsi l'effet curatif des virus oncolytiques dans le traitement des maladies hypoxiques, telles que les tumeurs solides.
PCT/CN2022/126657 2022-10-21 2022-10-21 Système de régulation de la réplication virale en réponse à un environnement hypoxique et son utilisation Ceased WO2024082263A1 (fr)

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