WO2024119387A1 - System for controlling target gene expression in response to hypoxic environment and use thereof - Google Patents

Abstract

The present invention provides a system for controlling target gene expression in response to a hypoxic environment and use thereof. The system comprises a gene encoding a hypoxia sensing unit and a gene encoding a target gene expression unit which are separated or fused to each other, wherein the gene encoding the hypoxia sensing unit comprises a gene encoding a hypoxia responsive element and a gene encoding an artificial transcription factor which are linked via a gene encoding a viral transcription promoter; the gene encoding the target gene expression unit comprises a gene encoding an artificial transcription factor recognition element and a target gene which are linked via the gene encoding the viral transcription promoter. The system efficiently expresses the artificial transcription factor by means of induction in a normal oxygen environment, and inhibits the expression thereof in the hypoxic environment, thus ensuring hypoxic sensitivity. The low-level expression of the artificial transcription factor in the hypoxic environment removes the inhibition of the target gene, and starts the transcription and expression of the target gene, thus realizing the targeted delivery of the target gene in the hypoxic environment, and avoiding serious toxic and side effects caused by systematic administration.

Description

A system for controlling target gene expression in response to hypoxic environment and its use Technical Field

The present invention belongs to the field of biomedicine technology, and specifically relates to a system for controlling the expression of a target gene in response to a hypoxic environment and its use in preparing a drug for treating hypoxic diseases, such as cancer.

Background technique

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 difficult to solve the problem of the universality of cell immunotherapy. In comparison, oncolytic viruses have the advantages of high killing efficiency, good targeting, small 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 cancer. For patients with advanced cancer, 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. At present, oncolytic virus therapy is receiving widespread attention and has a very broad prospect in the treatment of tumors.

At the beginning of the last century, oncolytic virus therapy began to be introduced into cancer treatment strategies. To date, 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.

However, in the development process of nearly 100 years, due to the limitations of oncolytic viruses themselves, the heterogeneity of tumor tissues and the complexity of tumor cells, although a number of oncolytic virus drugs have been launched, the therapeutic effect is minimal, and the effect of single administration is difficult to reach clinical expectations. Therefore, its application has begun to be questioned, which greatly limits the clinical application of oncolytic viruses in solid tumors. 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.

The huge challenge facing oncolytic virus therapy is not only how to find highly sensitive virus types and matching indications, but another challenge comes from the virus itself. As a heterologous pathogen in the body, 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.

The relatively hypoxic tumor microenvironment of solid tumors is an ideal target for gene therapy. 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. Studies have shown that under hypoxic conditions, the expression level of hypoxia-inducible factor-1 (HIF-1) produced in the cell nucleus increases, and its binding to target genes increases, promoting target gene transcription, thereby causing a series of cellular responses to hypoxia. 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.

Summary of the invention

The object of the present invention is to provide a system for controlling the transcription and expression of a target gene in response to a hypoxic environment. The system is induced by a hypoxia-sensitive artificial transcription factor to be efficiently expressed in a normoxic environment, and the expression is inhibited in a hypoxic environment, thereby ensuring the hypoxia sensitivity of the system; and the low-level expression of the artificial transcription factor induced in the hypoxic environment releases the inhibition of the target gene, thereby starting the efficient transcription and expression of the target gene, achieving targeted delivery of the target gene in a hypoxic environment, and avoiding serious toxic side effects caused by systemic administration. Through this transcription expression regulation system, the safety of the drug can be enhanced without affecting its effectiveness. The present invention also provides the application of the hypoxia-sensitive transcription expression regulation system, which can be used to prepare drugs for treating hypoxic diseases such as solid tumors by accessing different target genes.

On the one hand, the present invention provides a system for controlling the expression of a target gene in response to a hypoxic environment, which comprises a gene encoding a hypoxia sensing unit and a gene encoding a target gene expression unit, which are separated from or fused to each other, wherein the gene encoding the hypoxia sensing unit comprises a gene encoding a hypoxia response element (HRE) and a gene encoding an artificial transcription factor (ATF) connected via a gene encoding a virus transcription promoter (VTP), and the gene encoding the target gene expression unit comprises a gene encoding an artificial transcription factor recognition element (ATFRE) and a target gene (gene of interest, GOI) connected via a gene encoding a virus transcription promoter.

In the present invention, 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. As the blood supply to tumor tissues is insufficient, hypoxia of tumor cells and tissues occurs, and the oxygen content of cells and tissues is often less than 2%.

According to some embodiments of the present invention, a gene encoding a joint is included between the gene encoding VTP and the gene encoding HRE, and between the gene encoding VTP and the gene encoding ATF. The nucleotide sequence of the gene encoding the joint is a random sequence, generally 10-20 bp in size.

According to some embodiments of the present invention, a gene encoding a linker is included between the gene encoding VTP and the gene encoding ATFRE, and between the gene encoding VTP and GOI. The nucleotide sequence of the gene encoding the linker is a random sequence, generally 10-20 bp in size.

According to some embodiments of the present invention, the nucleotide sequence of the gene encoding HRE is as shown in SEQ ID NO: 1, or is a repeated nucleotide sequence of more than 1, preferably 3, and more preferably 5 nucleotide sequences as shown in SEQ ID NO: 1.

According to some embodiments of the present invention, the VTP is selected from one or more of PE/L, truncated PE/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), LTRminiP or P11.

According to some specific embodiments of the present invention, 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 nucleotide sequence of the P11 is as shown in SEQ ID NO: 37.

According to some embodiments of the present invention, the gene encoding ATF is selected from one or more of the CRE gene with a denuclearized localization signal (dNLS), the CRE-VP64 gene with a denuclearized localization signal, the CRE-VP64-ODD gene with a denuclearized localization signal, the CRE-RAP94 gene with a denuclearized localization signal, the CRE-RAP94-ODD gene with a denuclearized localization signal, or the CRE-VP160 gene with a denuclearized localization signal, and is preferably the CRE-VP64 gene with a denuclearized localization signal.

According to some specific embodiments of the present invention, the nucleotide sequence of the CRE gene with the denuclearization localization signal is shown in SEQ ID NO: 21 (the corresponding amino acid sequence is shown in SEQ ID NO: 22); the nucleotide sequence of the CRE-VP64 gene with the denuclearization localization signal is shown in SEQ ID NO: 23 (the corresponding amino acid sequence is shown in SEQ ID NO: 24); the nucleotide sequence of the CRE-VP64-ODD gene with the denuclearization localization signal is shown in SEQ ID NO: 25 (the corresponding amino acid sequence is shown in SEQ ID NO: 26 The nucleotide sequence of the CRE-RAP94 gene with the nuclear localization signal is shown in SEQ ID NO: 27 (the corresponding amino acid sequence is shown in SEQ ID NO: 28); the nucleotide sequence of the CRE-RAP94-ODD gene with the nuclear localization signal is shown in SEQ ID NO: 29 (the corresponding amino acid sequence is shown in SEQ ID NO: 30); the nucleotide sequence of the CRE-VP160 gene with the nuclear localization signal is shown in SEQ ID NO: 31 (the corresponding amino acid sequence is shown in SEQ ID NO: 32).

According to some embodiments of the present invention, the gene encoding ATFRE is one or more Loxp genes.

According to some specific embodiments of the present invention, the nucleotide sequence of the gene encoding ATFRE is a repeated gene sequence of more than 1, preferably 3, more preferably 5, and further preferably 6 Loxp genes, such as the nucleotide sequence shown in SEQ ID NO: 20.

According to some embodiments of the present invention, the GOI is selected from one or more of co-stimulatory molecules, cytokines, negative regulatory molecules, blocking antibodies of signal pathways, chemokines or killer molecules, preferably killer molecules, and more preferably one or more of the encoding genes of BiTE.

According to some specific embodiments of the present invention, the GOI is αCD47-αCD3-BiTE and/or αIGF1R-αCD3-BiTE.

According to some preferred embodiments of the present invention, the composition of the hypoxia sensing unit is 5*SEQ ID NO: 1-gene encoding VTP-dNLS-CRE-VP64 gene, and/or the composition of the target gene expression unit is SEQ ID NO: 20-gene encoding VTP-GOI. More preferably, the composition of the hypoxia sensing unit is 5*SEQ ID NO: 1-gene encoding PE/L-dNLS-CRE-VP64 gene, and/or the composition of the target gene expression unit is SEQ ID NO: 20-gene encoding P7.5-GOI. More preferably, the composition of the hypoxia sensing unit is 5*SEQ ID NO: 1-gene encoding PE/L-dNLS-CRE-VP64 gene, and/or the composition of the target gene expression unit is SEQ ID NO: 20-gene encoding P11-GOI.

According to some embodiments of the present invention, the system for controlling the expression of a target gene in response to a hypoxic environment according to the present invention comprises:

A first vector comprising the gene encoding the hypoxia sensing unit and a second vector comprising the gene encoding the target gene expression unit.

According to some embodiments of the present invention, the system for controlling the expression of a target gene in response to a hypoxic environment comprises a vector comprising a fused gene encoding the hypoxia sensing unit and the gene encoding the target gene expression unit.

According to some embodiments of the present invention, the vector is selected from one or more of pUC18, pUC19, pUC57, pcDNA3, pcDNA4, pcDNA5, pcDNA6, pCMV, pEF1, PEGFP, pET, pEasy, pAc5, pAcGP, pAcYCDuet, pBluescript, pBudCE, pCAMBIA, pCold, pCR2, pCR3, pCR4, pDsRED, pGEM, pGL, pIRES, pPIC, pMAL or pVRCSV1.0, etc.

In another aspect, the present invention provides a recombinant virus, the genome of which comprises the system for controlling the expression of a target gene in response to a hypoxic environment according to the present invention.

According to some embodiments of the present invention, the virus 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.

In another aspect, the present invention provides a pharmaceutical composition for treating hypoxic diseases, comprising a system for controlling the expression of a target gene in response to a hypoxic environment according to the present invention and a virus for treating a hypoxic disease, or a recombinant virus according to the present invention, and optional pharmaceutically acceptable excipients.

According to some embodiments of the present invention, the virus for treating hypoxic diseases 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.

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, 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.

According to some embodiments of the present invention, the pharmaceutical composition further comprises other active pharmaceutical ingredients for treating hypoxic diseases, such as PD-1 immune checkpoint inhibitors and/or CTLA-4 immune checkpoint inhibitors.

In another aspect, the present invention provides use of the system for controlling target gene expression in response to a hypoxic environment 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 medicament for treating hypoxic diseases.

According to some embodiments of the present invention, the hypoxic disease is cancer. Preferably, the cancer is leukemia and/or lymphoma. Preferably, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, sarcoma, thyroid cancer or brain cancer.

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

According to some embodiments of the present invention, the drug is an injection.

Accordingly, the present invention provides a method for treating hypoxic diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a system for controlling expression of a target gene in response to a hypoxic environment according to the present invention and a virus for treating a hypoxic disease, a recombinant virus according to the present invention, or a pharmaceutical composition according to the present invention.

According to some embodiments of the present invention, the hypoxic disease is cancer. Preferably, the cancer is leukemia and/or lymphoma. Preferably, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumor (GIST), liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, sarcoma, thyroid cancer or brain cancer.

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

The present invention provides a combination of molecular elements that can enable viruses to efficiently express exogenous genes under hypoxic conditions, and the expression is better than that under normoxic conditions, and its preparation method and application. The regulatory system comprising an artificial transcription factor that senses hypoxia can be applied to the treatment of hypoxic diseases such as solid tumors, especially oncolytic viruses for the treatment of solid tumors. The regulatory system comprising an artificial transcription factor that senses hypoxia consists of a hypoxia response element, a hypoxia-sensitive artificial transcription factor, an artificial transcription factor recognition element, and a target gene expression frame. The system has the following three advantages: 1) The artificial transcription factor that senses hypoxia can induce efficient expression in a normoxic environment, and inhibit expression in a hypoxic environment, thereby ensuring the hypoxia sensitivity of the system; 2) Inducing low-level expression of artificial transcription factors in a hypoxic environment, relieving the inhibition of the target gene, thereby starting the efficient transcription and expression of the target gene, thereby achieving targeted delivery in a hypoxic environment, and avoiding serious toxic side effects caused by systemic administration; 3) The protein encoded by the target gene has not been modified by any hypoxia-sensitive functional domain, which can ensure that the protein function encoded by the target gene is not disturbed and exerts its maximum activity.

It should be understood that within the scope of the present invention, the above-mentioned various technical features of the present invention and the various technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

Figures 1a-c show the hypoxic regulation of the HRE-VTP-GOI model. Figure 1a is a map of the plasmid loaded with HRE-PE/L-Luciferase-mCherry at the VGF2 gene site of vaccinia virus; Figure 1b is a map of the overexpression gene vector pHGAE plasmid loaded with dNLS-HIF-1α (WT)-P2A-eGFP. Using recombinant technology, the gene in the plasmid shown in Figure 1a was inserted into the vaccinia virus genome, and the cells were infected for 24 hours under normoxic (21% O ₂ ) and hypoxic (1% O ₂ ) conditions, or the gene in the plasmid shown in Figure 1b was inserted into the vaccinia virus genome to overexpress the dNLS-HIF-1α (WT) gene at the same time. It can be observed that compared with normoxic conditions, the expression of luciferase under hypoxic conditions is downregulated (Figure 1c).

Figure 2a-f shows maps of plasmids with HRE-VTP-ATF patterns of different ATF genes. Figure 2a shows a map of a plasmid (pHAGE-dNLS-CRE-P2A-eGFP) overexpressing a CRE gene with a denuclearization localization signal; Figure 2b shows a map of a plasmid (pHAGE-dNLS-CRE-VP64-P2A-eGFP) overexpressing a CRE-VP64 gene with a denuclearization localization signal; Figure 2c shows a map of a plasmid (pHAGE-dNLS-CRE-VP64-ODD-P2A-eGFP) overexpressing a CRE-VP64-ODD gene with a denuclearization localization signal; Figure 2d shows a map of a plasmid (pHAGE-dNLS-CRE-RAP94-P2A-eGFP) overexpressing a CRE-RAP94 gene with a denuclearization localization signal; Figure 2e shows a map of a plasmid (pHAGE-dNLS-CRE-RAP94-ODD-P2A-eGFP) overexpressing a CRE-RAP94-ODD gene with a denuclearization localization signal; Figure 2f shows a map of a plasmid (pHAGE-dNLS-CRE-RAP94-ODD-P2A-eGFP) overexpressing a CRE-RAP94-ODD gene with a denuclearization localization signal; Map of the plasmid containing the CRE-VP160 gene without the nuclear localization signal (pHAGE-dNLS-CRE-VP160-P2A-eGFP).

Figure 3a-g shows the map of the regulatory plasmid of the ATFRE-VTP-GOI model. Figure 3a shows the map of the plasmid loaded with 6*Loxp-LTRminiP-Luciferase-mCherry at the VGF2 gene site of vaccinia virus; Figure 3b shows the map of the plasmid loaded with 6*Loxp-PE/L-S-Luciferase-mCherry at the VGF2 gene site of vaccinia virus; Figure 3c shows the map of the plasmid loaded with 6*Loxp-PE/L-L-Luciferase-mCherry at the VGF2 gene site of vaccinia virus; Figure 3d shows the map of the plasmid loaded with 6*Loxp- Figure 3e shows a map of a plasmid loaded with 6*Loxp-P7.5-24-Luciferase-mCherry at the vaccinia virus VGF2 gene site; Figure 3f shows a map of a plasmid loaded with 6*Loxp-P7.5-71-Luciferase-mCherry at the vaccinia virus VGF2 gene site; Figure 3g shows a map of a plasmid loaded with 6*Loxp-P7.5-Luciferase-mCherry at the vaccinia virus VGF2 gene site.

Figures 4a and b show that loading ATF can inhibit the expression of the target gene using the ATFRE-VTP-GOI model. The genes in the plasmid shown in Figure 3 were inserted into the vaccinia virus genome using recombination technology, and then the ATF gene in the plasmid shown in Figure 2 was overexpressed, and downregulation of luciferase expression was observed, where Blank represents a blank control without transfection of the plasmid shown in Figure 2.

Figure 5a-d shows that HRE-VTP-ATF and ATFRE-VTP-GOI can mediate the virus to specifically express the target gene under hypoxic conditions. Among them, Figure 5a shows the map of the plasmid loaded with 6*Loxp-P7.5-αCD47-αCD3-BiTE at the vaccinia virus C9 gene site; Figures 5b and 5c show the maps of the plasmid integrated with HRE-PE/L-L-dNLS-CRE-RAP94-ODD at the vaccinia virus C16 and VGF1 gene sites, respectively; Figure 5d shows that 6*Loxp-P7.5-αCD47-αCD3-BiTE and HRE-PE/L-L-dNLS-CRE-RAP94-ODD were inserted into the vaccinia virus genome by recombination technology, and SKOV3 tumor cells were infected under normoxic and hypoxic conditions for 24 hours. It can be observed that the amount of killing factor αCD47-αCD3-BiTE released by the virus under hypoxic conditions is significantly higher than the amount released under normoxic conditions, that is, SKOV3 tumor cells are selectively killed under hypoxic environment.

Figure 6a-g shows that HRE-VTP-ATF and ATFRE-VTP-GOI can mediate the specific expression of target genes under hypoxic conditions. Among them, Figure 6a-d shows the quality of vaccinia virus C9 gene locus loaded with 6*Loxp-P7.5-αCD47-αCD3-BiTE, 6*Loxp-P11-αCD47-αCD3-BiTE, 6*Loxp-P7.5-αIGF1R-αCD3-BiTE and 6*Loxp-P11-αIGF1R-αCD3-BiTE Figure 6e shows a map of the plasmid integrating HRE-PE/L-L-dNLS-CRE-VP64 at the vaccinia virus C16 gene locus; Figures 6f and 6g show the use of recombination technology to integrate 6*Loxp-P7.5-αCD47-αCD3-BiTE and HRE-PE/L-L-dNLS-CRE-VP64, 6*Loxp-P11-αCD47-αCD3-BiTE and HRE-PE/L-L-dNLS-CRE-VP64, 6*Loxp-P7.5-αIGF1R-αCD3-BiTE, respectively. E and HRE-PE/L-L-dNLS-CRE-VP64, as well as 6*Loxp-P11-αIGF1R-αCD3-BiTE and HRE-PE/L-L-dNLS-CRE-VP64 were loaded into the vaccinia virus genome, and SKOV3 cells were infected under normoxic and hypoxic conditions for 24 hours. It was observed that the amount of killing factors αCD47-αCD3-BiTE or αIGF1R-αCD3-BiTE released by the virus under hypoxic conditions was significantly higher than that released under normoxic conditions, that is, SKOV3 tumor cells were selectively killed under hypoxic conditions.

Figure 7 shows that intratumorally injected hypoxia-sensitive transcriptional expression control system-controlled oncolytic viruses (TTV-P7.5-347-CRE-VP64 and TTV-P11-347-CRE-VP64) can effectively inhibit tumor growth. Among them, Figure 7a shows the inhibition of NCI-H292 tumor cells; Figure 7b shows the inhibition of SKOV3 tumor cells; TTV: Tiantan vaccinia virus, vaccinia virus Tiantan strain; UTD: Untransduced, that is, cytokine-induced cultured but not transduced T cells.

Best Mode for Carrying Out the Invention

The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer with these descriptions. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

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. Among them, 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. Severe combined immunodeficiency mice (B-NDG) were purchased from Shanghai South Model Organisms Technology Co., Ltd.

Example 1 Construction of vaccinia virus recombinant plasmid and artificial transcription factor overexpression plasmid

The nucleotide sequence shown in SEQ ID NO: 11 (the corresponding amino acid sequence is shown in SEQ ID NO: 12) was synthesized by Shanghai Jierui Biotechnology Co., Ltd. and cloned into the pHAGE expression vector to obtain the pHAGE-dNLS-HIF-1α (WT)-P2A-eGFP overexpression plasmid, and its plasmid map is shown in Figure 1b.

The nucleotide sequences shown in SEQ ID NO: 13-19 were synthesized by Shanghai Jierui Bioengineering Co., Ltd. and cloned into the VGF2 gene recombinant plasmid respectively to obtain the 6*Loxp-PE/L-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 13; the 6*Loxp-PE/L-L-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 14; the 6*Loxp-PE/L-S-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 15; and the 6*Loxp-P 7.5-Luciferase-mCherry recombinant plasmid; 6*Loxp-P7.5-71-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 17; 6*Loxp-P7.5-24-Luciferase-mCherry recombinant plasmid carrying the nucleotide sequence shown in SEQ ID NO: 18; and 6*Loxp-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 3a-g.

The nucleotide sequences shown in SEQ ID NO: 21, 23, 25, 27, 29 and 31 were synthesized by Shanghai Jierui Biotechnology Co., Ltd. and cloned into the pHAGE expression vector respectively to obtain the pHAGE-dNLS-CRE-P2A-eGFP overexpression plasmid carrying the nucleotide sequence shown in SEQ ID NO: 21; the pHAGE-dNLS-CRE-VP64-P2A-eGFP overexpression plasmid carrying the nucleotide sequence shown in SEQ ID NO: 23; and the pHAGE-dNLS-CRE-VP64-P2A-eGFP overexpression plasmid carrying the nucleotide sequence shown in SEQ ID NO: 25. LS-CRE-VP64-ODD-P2A-eGFP overexpression plasmid; pHAGE-dNLS-CRE-RAP94-P2A-eGFP overexpression plasmid carrying the nucleotide sequence shown in SEQ ID NO: 27; pHAGE-dNLS-CRE-RAP94-ODD-P2A-eGFP overexpression plasmid carrying the nucleotide sequence shown in SEQ ID NO: 29; and pHAGE-dNLS-CRE-VP160-P2A-eGFP overexpression plasmid carrying the nucleotide sequence shown in SEQ ID NO: 31. The above overexpression plasmids all use green fluorescence signals as recombination screening signals, and their plasmid maps are shown in Figures 2a-f.

The nucleotide sequences shown in SEQ ID NO: 1, 3 and 9 were synthesized by Shanghai Jierui Bioengineering Co., Ltd., spliced together by homologous recombination, and cloned into the VGF-2 gene recombinant plasmid to obtain the pSC65-VGF-2-HRE-PE/L-L-Luciferase-mCherry recombinant plasmid carrying the HRE-PE/L-L-Luciferase gene. The recombinant plasmid uses a red fluorescent signal as a recombination screening signal, and its plasmid map is shown in Figure 1a.

The nucleotide sequences shown in SEQ ID NOs: 20, 5 and 33 (the amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 33 is shown in SEQ ID NO: 34) were synthesized by Shanghai Jierui Bioengineering Co., Ltd., spliced together by homologous recombination, and cloned into the C9 gene recombination plasmid to obtain the pSC65-C9-6*Loxp-P7.5-αCD47-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P7.5-αCD47-αCD3-BiTE gene (see Figures 5a and 6a). The recombinant plasmid uses a blue fluorescent signal as a recombination screening signal. The nucleotide sequences shown in SEQ ID NO: 20, 37 and 33 were synthesized, spliced together by homologous recombination, and cloned into the C9 gene recombination plasmid to obtain the pSC65-C9-6*Loxp-P11-αCD47-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P11-αCD47-αCD3-BiTE gene (see Figure 6b). The recombinant plasmid uses a blue fluorescent signal as a recombination screening signal.

The nucleotide sequences shown in SEQ ID NOs: 20, 5 and 35 (the amino acid sequence corresponding to the nucleotide sequence shown in SEQ ID NO: 35 is shown in SEQ ID NO: 36) were synthesized by Shanghai Jierui Biotechnology Co., Ltd., spliced together by homologous recombination, and cloned into the C9 gene recombination plasmid to obtain the pSC65-C9-6*Loxp-P7.5-αIGF1R-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P7.5-αIGF1R-αCD3-BiTE gene. The nucleotide sequences shown in SEQ ID NOs: 20, 37 and 35 were synthesized, spliced together by homologous recombination, and cloned into the C9 gene recombination plasmid to obtain the pSC65-C9-6*Loxp-P11-αIGF1R-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P11-αIGF1R-αCD3-BiTE gene (see Figure 6d), which used a blue fluorescence signal as a recombination screening signal.

The nucleotide sequences shown in SEQ ID NO: 1, 3 and 29 were synthesized by Shanghai Jierui Biotechnology Co., Ltd., spliced together by homologous recombination, and cloned into C16 and VGF gene recombinant plasmids, respectively, to obtain pSC65-C16-HRE-PE/L-L-dNLS-CRE-RAP94-ODD-eGFP recombinant plasmid and pSC65-VGF-HRE-PE/L-L-dNLS-CRE-RAP94-ODD-eGFP recombinant plasmid carrying HRE-PE/L-L-dNLS-CRE-RAP94-ODD gene (see Figure 5b and c), and the above recombinant plasmids used green fluorescence signal as the recombination screening signal; Synthesized SEQ ID NO: The nucleotide sequences shown in 1, 3 and 23 were spliced together by homologous recombination and cloned into the C16 gene recombination plasmid to obtain the pSC65-C16-HRE-PE/L-L-dNLS-CRE-VP64-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-VP64 gene (see Figure 6e).

Example 2 Recombination, purification and titer determination of vaccinia virus

1. Recombination of vaccinia virus using vaccinia gene recombinant plasmid

1.1 Cell preparation: 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.

1.2 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 medium (DMEM medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin antibiotics (PS)).

1.3 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.

1.4 Prepare 2×DMEM maintenance medium (DMEM medium + 2% FBS + 1% PS) for virus plating and add 2% preheated low-melting point agarose.

1.5 Aspirate the supernatant in the 6-well plate, add 4 mL of the mixture for plating into the 6-well plate, 300 μL per well. Then carefully place it in a 4°C refrigerator to promote solidification, and transfer it to a 37°C incubator for culture after the low melting point agarose solidifies.

1.6 Pick the recombinant virus plaques under a fluorescence microscope and add 500 μL of DMEM complete medium. Repeat freezing and thawing at -80°C for more than three times to release as much virus as possible. This purification process needs to be performed at least 5 times.

1.7 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.

1.8 Before inoculating the virus, replace the culture medium in the well with 2 mL DMEM maintenance medium. Repeatedly blow the purified virus solution containing fluorescence until it is dispersed. Add about 100 μL of virus solution to each well. Incubate in a 37°C incubator for about 48 hours, and collect samples according to the formation of virus plaques.

1.9 Sample collection: Carefully aspirate 1 mL of the culture medium supernatant in the well. Use the remaining 1 mL of culture medium to fully blow off the cells and collect them in an EP tube, which can be used for subsequent genome extraction and as a virus seed for amplification.

2. Amplification and Purification of Recombinant Vaccinia Virus

2.1 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;

2.2 Before virus inoculation, replace DMEM complete medium with 8 mL DMEM maintenance medium, inoculate the virus into the cells in DMEM maintenance medium, and the inoculation amount is about 0.02 MOI (MOI = virus PFU/cell number). Continue to culture in an incubator at 37°C and 5% _CO2 for about 48 hours, and collect samples according to the formation of virus plaques;

2.3 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;

2.4 After 24 hours of freezing, freeze-thaw the collected virus solution twice, perform density gradient centrifugation with 36% sucrose solution, centrifuge at 16000g and 4°C for 90 minutes, carefully pour out the supernatant, dissolve the virus precipitate in the centrifuge tube with PBS buffer, and store it in aliquots at -80°C until the virus titer is determined.

3. Titer determination of recombinant vaccinia virus

3.1 Preparation of ^TK143 cells: ^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;

3.2 Dilute the virus. Use DMEM to dilute the vaccinia virus solution, starting from 1:100, and make 10-fold dilutions to a final volume of 1100 μL.

3.3 Discard the DMEM complete medium in the 24-well plate, take 500 μL of the diluted virus solution and add it to the wells, and make two duplicate wells. Incubate in an incubator at 37°C and 5% CO ₂ for about 48 hours, and determine the plaque spreading time according to the formation of virus plaques;

3.4 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.

Example 3 Hypoxia-regulated effects of the HRE-VTP-GOI model in vaccinia virus

In this example, vaccinia virus infection experiments were conducted in ^TK143- cells under normoxic and hypoxic conditions to test the expression of target genes by vaccinia viruses in which a hypoxia-sensitive transcriptional expression control system was integrated into the viral genome.

1. Figure 1a shows the map of the plasmid carrying HRE-PE/L-L-Luciferase-mCherry at the vaccinia virus VGF2 gene site constructed in Example 1. Using recombinant technology, the target gene in the plasmid of Figure 1a was inserted into the vaccinia virus genome using the vaccinia virus recombinant plasmid and the method of Example 2, and verified.

2. TK143 ^- cells were plated into 96-well plates at 1× ¹⁰⁴ cells/well. The next day, the cells were attached to the wall and divided into two groups. One group was transfected with the overexpression plasmid of pHAGE-dNLS-HIF-1α(WT)-P2A-eGFP (200ng per well), and the other group was a blank control group (Blank). 24 hours after plasmid transfection, the cells were infected with the recombinant vaccinia virus obtained in step 1, and the multiplicity of infection (MOI) was 0.02. After infection, the cells were placed in hypoxic conditions (1% _O2 concentration) and normoxic conditions (21% _O2 concentration) for 24 hours. After the culture, the cells were collected and the expression of luciferase was detected.

微孔板发光检测仪检测靶细胞的荧光素酶活力值，根据图1c所示的结果，缺氧感应模式HRE-VTP-GOI在缺氧条件下可以显著抑制目的基因表达，Blank组和过表达HIF-1α组分别有31.8和50.4倍的下调，表明过表达HIF-1α蛋白进一步增加了对目的基因表达的抑制。 use

The luciferase activity value of the target cells was detected by a microplate luminescence detector. According to the results shown in Figure 1c, the hypoxia sensing mode HRE-VTP-GOI can significantly inhibit the expression of the target gene under hypoxic conditions. The Blank group and the HIF-1α overexpression group had 31.8 and 50.4-fold downregulation, respectively, indicating that overexpression of HIF-1α protein further increased the inhibition of target gene expression.

Example 4 Different ATFs in vaccinia virus can inhibit the expression of the target gene in ATFRE-VTP-GOI

In this example, a vaccinia virus infection experiment under hypoxic conditions was conducted in ^TK143- cells to test the expression of a target gene by a vaccinia virus in which a hypoxia-sensitive transcriptional expression control system was integrated into the viral genome.

1. Using recombinant technology, the plasmid constructed in Example 1 with 6*Loxp-LTRminiP-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3 a ), the plasmid with 6*Loxp-PE/L-S-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3 b ), the plasmid with 6*Loxp-PE/L-L-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3 c ), and the plasmid with 6*Loxp-PE/L-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3 e ) were used. The target genes were inserted into the vaccinia virus genome by using a plasmid containing erase-mCherry ( FIG. 3d ), a plasmid containing 6*Loxp-P7.5-24-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3e ), a plasmid containing 6*Loxp-P7.5-71-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3f ), and a plasmid containing 6*Loxp-P7.5-Luciferase-mCherry at the vaccinia virus VGF2 gene site ( FIG. 3g ) and the method of Example 2, respectively, and verified.

2. TK143 ^- cells were plated into 96-well plates at 1× ¹⁰⁴ cells/well. After the cells adhered to the wall the next day, the plasmids shown in Figure 2 were transfected (200 ng per well). 24 hours after plasmid transfection, the recombinant vaccinia virus obtained in step 1 was infected, and the virus infection multiplicity (MOI) was 0.02. After infection, the cells were cultured for another 24 hours. After the culture was completed, the cells were collected and the expression of luciferase was detected.

微孔板发光检测仪检测靶细胞的荧光素酶活力值，根据图4a和4b所示，不同病毒启动子控制下，不同种ATF均可抑制ATFRE-VTP-GOI模式中的目的基因的表达，不同病毒的启动子抑制荧光素酶表达的下降倍数在5-10倍之间。 3. Use

The luciferase activity value of the target cells was detected by a microplate luminescence detector. As shown in Figures 4a and 4b, different types of ATFs under the control of different viral promoters could inhibit the expression of the target gene in the ATFRE-VTP-GOI model, and the reduction factor of luciferase expression inhibited by different viral promoters was between 5-10 times.

Example 5 The hypoxia-sensitive transcriptional expression regulatory system can mediate the specific expression of the target gene by vaccinia virus under hypoxic conditions at different integration sites

In this example, vaccinia virus infection experiments were conducted in ^TK143- cells under normoxic and hypoxic conditions to test the expression of target genes by vaccinia viruses with hypoxia-sensitive transcriptional expression regulatory systems at different integration sites of the viral genome.

1. The pSC65-C16-HRE-PE/L-L-dNLS-CRE-RAP94-ODD-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-RAP94-ODD gene and the pSC65-C9-6*Loxp-P7.5-αCD47-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P7.5-αCD47-αCD3-BiTE gene constructed in Example 1 and the method of Example 2 were used to recombinant vaccinia virus and verified.

2. The pSC65-VGF-HRE-PE/L-L-dNLS-CRE-RAP94-ODD-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-RAP94-ODD gene and the pSC65-C9-6*Loxp-P7.5-αCD47-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P7.5-αCD47-αCD3-BiTE gene constructed in Example 1 and the method of Example 2 were used to recombinant vaccinia virus and verified.

3. TK143 ^- cells were plated into 12-well plates at 2×10 ⁵ cells/well, and infected after the cells adhered to the wall the next day. The virus infection multiplicity (MOI) was 0.02, and the cells were infected with vaccinia virus with HRE-PE/LL-dNLS-CRE-RAP94-ODD and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated at the C16 and C9 gene loci; and infected with vaccinia virus with HRE-PE/LL-dNLS-CRE-RAP94-ODD and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated at the VGF and C9 gene loci. After infection, the cells were cultured under hypoxic conditions (1% O ₂ concentration) and normoxic conditions (21% O ₂ concentration) for 24 hours, and the cell supernatant was collected after the culture to detect the expression of killing factors.

微孔板发光检测仪检测靶细胞的荧光素酶活力值。 4. The tumor cell killing efficiency was evaluated by luciferase-based cytotoxicity assay. First, 1×10 ⁴ SKOV3-Luc (human ovarian cancer cells modified by firefly luciferase gene) were inoculated on a 96-well flat-bottom black plate, 100 μL culture medium per well, and cultured in a 37°C, 5% CO ₂ cell culture incubator for 18 hours. On the second day, the culture supernatant obtained in step 3 was added to the corresponding wells, and the co-culture cell supernatant (control group) was set up. After the culture was completed, the supernatant was used.

The luciferase activity of target cells was detected by a microplate luminescence detector.

The calculation formula of cell killing rate is as follows:

Cell killing rate (%) = (luciferase activity value of virus-infected supernatant in control group - luciferase activity value of experimental group) / luciferase activity value of virus-infected supernatant in control group × 100

5. The results are shown in Figure 5d. The oncolytic virus with HRE-PE/L-L-dNLS-CRE-RAP94-ODD and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated into different gene sites C16/VGF and C9 of vaccinia virus respectively had low tumor killing activity under normoxic conditions, with killing rates of only 10.4% and 5.04%, respectively, but could selectively and efficiently kill SKOV3 tumor cells under hypoxic conditions (killing rates were 17.7% and 12%, respectively), that is, the killing activity of specific tumor cell killing under hypoxic conditions was 1.7 times and 2.4 times that under normoxic conditions, respectively.

Example 6 In vitro antitumor effect of oncolytic virus controlled by hypoxia-sensitive transcriptional expression regulatory system

In this example, vaccinia virus infection experiments were conducted in ^TK143- cells under normoxic and hypoxic conditions to test the expression of target genes by viruses with hypoxia-sensitive transcriptional expression regulatory systems.

1. The pSC65-C16-HRE-PE/L-L-dNLS-CRE-VP64-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-VP64 gene and the pSC65-C9-6*Loxp-P7.5-αCD47-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P7.5-αCD47-αCD3-BiTE gene constructed in Example 1 and the method of Example 2 were used to recombinant vaccinia virus and verified; The vaccinia virus was recombined using the pSC65-C16-HRE-PE/L-L-dNLS-CRE-VP64-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-VP64 gene constructed in Example 1 and the pSC65-C9-6*Loxp-P11-αCD47-αCD3-BiTE-BFP recombinant plasmid carrying the nucleic acid sequence 6*Loxp-P11-αCD47-αCD3-BiTE gene and the method of Example 2, and the results were verified.

2. The pSC65-C16-HRE-PE/L-L-dNLS-CRE-VP64-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-VP64 gene constructed in Example 1 and the The vaccinia virus was recombined using the pSC65-C9-6*Loxp-P7.5-αIGF1R-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P7.5-αIGF1R-αCD3-BiTE gene and the method of Example 2, and the results were verified. The vaccinia virus was recombined using the pSC65-C16-HRE-PE/L-L-dNLS-CRE-VP64-eGFP recombinant plasmid carrying the HRE-PE/L-L-dNLS-CRE-VP64 gene constructed in Example 1 and the pSC65-C9-6*Loxp-P11-αIGF1R-αCD3-BiTE-BFP recombinant plasmid carrying the 6*Loxp-P11-αIGF1R-αCD3-BiTE gene and the method of Example 2, and the results were verified.

3. Plate ^TK143- cells at 2× ¹⁰⁵ cells/well into a 12-well plate and infect them after the cells adhere to the plate the next day. The virus infection multiplicity (MOI) was 0.02, and the cells were infected with vaccinia virus with HRE-PE/LL-dNLS-CRE-VP64 and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated at the C16 and C9 gene loci, vaccinia virus with HRE-PE/LL-dNLS-CRE-VP64 and 6*Loxp-P11-αCD47-αCD3-BiTE genes integrated at the C16 and C9 gene loci, vaccinia virus with HRE-PE/LL-dNLS-CRE-VP64 and 6*Loxp-P7.5-αIGF1R-αCD3-BiTE genes integrated at the C16 and C9 gene loci, and vaccinia virus with HRE-PE/LL-dNLS-CRE-VP64 and 6*Loxp-P11-αIGF1R-αCD3-BiTE genes integrated at the C16 and C9 gene loci. After infection, the cells were cultured under hypoxic conditions (1% O ₂ concentration) and normoxic conditions (21% O ₂ concentration) for 24 h. After the culture, the cell supernatant was collected to detect the expression of the killing factor.

微孔板发光检测仪检测靶细胞的荧光素酶活力值。 4. The tumor cell killing efficiency was evaluated by luciferase-based cytotoxicity assay. First, 1×10 ⁴ SKOV3-Luc (human ovarian cancer cells modified by firefly luciferase gene) were inoculated on a 96-well flat-bottom black plate, 100 μL culture medium per well, and cultured in a 37°C, 5% CO ₂ cell culture incubator for 18 hours. On the second day, the culture supernatant obtained in step 3 was added to the corresponding wells, and the co-culture cell supernatant (control group) was set up. After the culture was completed, the supernatant was used.

The luciferase activity of target cells was detected by a microplate luminescence detector.

The calculation formula of cell killing rate is as follows:

Cell killing rate (%) = (luciferase activity value of virus-infected supernatant in control group - luciferase activity value of experimental group) / luciferase activity value of virus-infected supernatant in control group × 100

5. The results are shown in Figures 6f and 6g. Figure 6f shows vaccinia virus with HRE-PE/L-L-dNLS-CRE-VP64 and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated into the C16 and C9 gene loci, and vaccinia virus with HRE-PE/L-L-dNLS-CRE-VP64 and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated into the C16 and C9 gene loci. The tumor killing activity of vaccinia virus carrying 6*Loxp-P11-αCD47-αCD3-BiTE gene was low under normoxic conditions, with killing rates of 18.2% and 13.8%, respectively. However, it could selectively and efficiently kill SKOV3 tumor cells under hypoxia (killing rates of 30.9% and 27.2%, respectively), that is, the killing activity of specific tumor cell killing under hypoxia was 1.7 times and 2.1 times that under normoxic conditions, respectively; Figure 6g shows that HRE-PE/L-L-dNLS-CRE-VP64 and 6*Loxp-P7.5-αIGF were integrated at the C16 and C9 gene loci. The vaccinia virus with the 1R-αCD3-BiTE gene and the vaccinia virus with the HRE-PE/L-L-dNLS-CRE-VP64 and 6*Loxp-P11-αIGF1R-αCD3-BiTE genes integrated at the C16 and C9 gene loci had low tumor killing activity under normoxic conditions, with killing rates of 5.3% and 6.2%, respectively. However, they could selectively and efficiently kill SKOV3 tumor cells under hypoxic conditions (killing rates were 21.5% and 18.5%, respectively), that is, the killing activity of specific tumor cell killing under hypoxic conditions was 3.1 times and 4.1 times that under normoxic conditions, respectively.

Example 7 In vivo anti-tumor effect of oncolytic virus controlled by hypoxia-sensitive transcriptional expression control system

One day in advance, perform local hair removal on the back of B-NDG mice housed in a sterile isolator. Hair removal can be done using a depilatory cream or an animal shaver to expose the skin at the site of tumor cell inoculation.

Fix the mouse with the left hand, that is, grasp the mouse's head, neck and back skin with the left hand at the same time, turn the mouse's back to the left, fully expose the shaved area on the right side of the back, and disinfect it with an alcohol cotton ball in the right hand. After blowing and mixing the pre-prepared SKOV3 or NCI-H292 tumor cells with a 1mL insulin syringe, draw 125μL of cell suspension (5×10 ⁶ tumor cells), insert the needle obliquely into the mouse's subcutaneous tissue at an angle of 30°-40° between the needle tip and the skin, and slowly push the cell suspension to avoid cell leakage. After the injection of 125μL of cell suspension is completed, leave the needle in for 2-3 seconds and then quickly pull it out. A clearly visible small bag can be 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 tumor formation, the baseline tumor volume was measured with a vernier caliper and subsequent experiments were performed.

5×10 ³ PFU of vaccinia virus with hypoxia-sensitive regulatory system, i.e., vaccinia virus with HRE-PE/LL-dNLS-CRE-VP64 and 6*Loxp-P7.5-αCD47-αCD3-BiTE genes integrated into C16 and C9 sites (TTV-P7.5-347-CRE-VP64), vaccinia virus with HRE-PE/LL-dNLS-CRE-VP64 and 6*Loxp-P11-αCD47-αCD3-BiTE genes integrated into C16 and C9 sites (TTV-P11-347-CRE-VP64) and control wild-type vaccinia virus were reinfused intratumorally. The tumor size was measured every 2-3 days. The long and short diameters of the tumor were measured with a vernier caliper. The tumor volume was calculated as follows:

Volume = (longer diameter × shorter ^diameter2 )/2.

The results are shown in Figure 7. The vaccinia viruses controlled by the hypoxia-sensitive transcriptional expression control system (TTV-P7.5-347-CRE-VP64 and TTV-P11-347-CRE-VP64) can effectively inhibit the growth of SKOV3 (ovarian cancer) and NCI-H292 (lung cancer). The tumor inhibition rates 66 days after virus injection were as high as 100% and 80%, respectively. Compared with the wild-type control group of vaccinia virus, 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-P7.5-347-CRE-VP64 and TTV-P11-347-CRE-VP64 groups were still alive at 60 days.

The above description is only the preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the profession can make some changes or modifications to the technical content disclosed above without departing from the scope of the technical solution of the present invention to obtain an equivalent embodiment with equivalent changes. However, any modification, equivalent change and modification made to the above embodiment 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 (12)

A system for controlling the expression of a target gene in response to a hypoxic environment, comprising a gene encoding a hypoxia sensing unit and a gene encoding a target gene expression unit, which are separated or fused from each other, wherein the gene encoding the hypoxia sensing unit comprises a gene encoding a hypoxia response element and a gene encoding an artificial transcription factor connected via a gene encoding a viral transcription promoter, and the gene encoding the target gene expression unit comprises a gene encoding an artificial transcription factor recognition element and a target gene connected via a gene encoding a viral transcription promoter. The system according to claim 1, wherein a gene encoding a linker is included between the gene encoding the viral transcription promoter and the gene encoding the hypoxia response element, and between the gene encoding the viral transcription promoter and the gene encoding the artificial transcription factor; and/or A gene encoding a linker is included between the gene encoding the viral transcription promoter and the gene encoding the artificial transcription factor recognition element, and between the gene encoding the viral transcription promoter and the target gene. A system according to claim 1 or 2, wherein the nucleotide sequence of the gene encoding the hypoxia response element is as shown in SEQ ID NO: 1, or is a repeated nucleotide sequence of more than 1, preferably 3, and more preferably 5 nucleotide sequences as shown in SEQ ID NO: 1. The system according to any one of claims 1 to 3, wherein the viral transcription promoter is selected from one or more of PE/L, truncated PE/L, further truncated PE/L, P7.5, truncated P7.5, further truncated P7.5, LTRminiP or P11; Preferably, 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 further 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 further truncated P7.5 is shown in SEQ ID NO: 7; the nucleotide sequence of the LTRminiP is shown in SEQ ID NO: 8; the nucleotide sequence of the P11 is shown in SEQ ID NO: 37; The system according to any one of claims 1 to 4, wherein the gene encoding the artificial transcription factor is selected from one or more of the CRE gene without a nuclear localization signal, the CRE-VP64 gene without a nuclear localization signal, the CRE-VP64-ODD gene without a nuclear localization signal, the CRE-RAP94 gene without a nuclear localization signal, the CRE-RAP94-ODD gene without a nuclear localization signal or the CRE-VP160 gene without a nuclear localization signal, preferably the CRE-VP64 gene without a nuclear localization signal; Further preferably, the nucleotide sequence of the CRE gene of the nuclear localization signal is shown as SEQ ID NO: 21; the nucleotide sequence of the CRE-VP64 gene of the nuclear localization signal is shown as SEQ ID NO: 23; the nucleotide sequence of the CRE-VP64-ODD gene of the nuclear localization signal is shown as SEQ ID NO: 25; the nucleotide sequence of the CRE-RAP94 gene of the nuclear localization signal is shown as SEQ ID NO: 27; the nucleotide sequence of the CRE-RAP94-ODD gene of the nuclear localization signal is shown as SEQ ID NO: 29; the nucleotide sequence of the CRE-VP160 gene of the nuclear localization signal is shown as SEQ ID NO: 31; The system according to any one of claims 1 to 5, wherein the gene encoding the artificial transcription factor recognition element is one or more Loxp genes; Preferably, the nucleotide sequence of the gene encoding the artificial transcription factor recognition element is a repeated gene sequence of more than 1, preferably 3, more preferably 5, and further preferably 6 Loxp genes, such as the nucleotide sequence shown in SEQ ID NO: 20. The system according to any one of claims 1 to 6, wherein the target gene is selected from one or more of co-stimulatory molecules, cytokines, negative regulatory molecules, blocking antibodies of signal pathways, chemokines or killer molecules, preferably killer molecules, more preferably one or more of the encoding genes of BiTE; preferably, the target gene is αCD47-αCD3-BiTE and/or αIGF1R-αCD3-BiTE. The system according to any one of claims 1 to 7, wherein the hypoxia sensing unit is composed of 5 repeated nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 1-a gene encoding a viral transcription promoter-a CRE-VP64 gene without a nuclear localization signal, and/or the target gene expression unit is composed of the nucleotide sequence shown in SEQ ID NO: 20-a gene encoding a viral transcription promoter-a target gene; Preferably, the hypoxia sensing unit is composed of 5 repeated nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 1 - a gene encoding PE/L - a CRE-VP64 gene without a nuclear localization signal, and/or the target gene expression unit is composed of the nucleotide sequence shown in SEQ ID NO: 20 - a gene encoding P7.5 - a target gene; Preferably, the hypoxia sensing unit is composed of 5 repeated nucleotide sequences of the nucleotide sequence shown in SEQ ID NO: 1 - a gene encoding PE/L - a CRE-VP64 gene with a nuclear localization signal, and/or the target gene expression unit is composed of the nucleotide sequence shown in SEQ ID NO: 20 - a gene encoding P11 - a target gene. The system according to any one of claims 1 to 8, wherein the system comprises: A first vector comprising the gene encoding the hypoxia sensing unit and a second vector comprising the gene encoding the target gene expression unit; or The system comprises a vector, wherein the vector comprises a fused gene encoding the hypoxia sensing unit and a gene encoding the target gene expression unit; Preferably, the vector is selected from one or more of pUC18, pUC19, pUC57, pcDNA3, pcDNA4, pcDNA5, pcDNA6, pCMV, pEF1, PEGFP, pET, pEasy, pAc5, pAcGP, pAcYCDuet, pBluescript, pBudCE, pCAMBIA, pCold, pCR2, pCR3, pCR4, pDsRED, pGEM, pGL, pIRES, pPIC, pMAL or pVRCSV1.0. A recombinant virus, the genome of which comprises the system according to any one of claims 1 to 9, Preferably, the virus is selected from one or more of vaccinia virus, adenovirus, herpes simplex virus type I, 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, lentivirus or retrovirus. A pharmaceutical composition for treating a hypoxic disease, comprising the system according to any one of claims 1 to 9 and a virus for treating a hypoxic disease, or the recombinant virus according to claim 10, and optional pharmaceutically acceptable excipients; Preferably, the virus for treating hypoxic diseases is selected from one or more of vaccinia virus, adenovirus, herpes simplex virus type I, 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, lentivirus or retrovirus; Preferably, 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; Preferably, the pharmaceutical composition further comprises other active pharmaceutical ingredients for treating hypoxic diseases, such as PD-1 immune checkpoint inhibitors and/or CTLA-4 immune checkpoint inhibitors. Use of the system according to any one of claims 1 to 9, the recombinant virus according to claim 10 or the pharmaceutical composition according to claim 11 in the preparation of a medicament for treating hypoxic diseases; Preferably, the hypoxic disease is cancer; further preferably, the cancer is leukemia and/or lymphoma; further preferably, the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, gastrointestinal stromal tumor, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, sarcoma, thyroid cancer or brain cancer; Preferably, the drug is an oncolytic virus drug. Preferably, the drug is an injection.

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