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WO2020238427A1 - Système de virus oncolytique pour tuer spécifiquement des cellules tumorales, et son application - Google Patents

Système de virus oncolytique pour tuer spécifiquement des cellules tumorales, et son application Download PDF

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WO2020238427A1
WO2020238427A1 PCT/CN2020/083823 CN2020083823W WO2020238427A1 WO 2020238427 A1 WO2020238427 A1 WO 2020238427A1 CN 2020083823 W CN2020083823 W CN 2020083823W WO 2020238427 A1 WO2020238427 A1 WO 2020238427A1
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nucleic acid
acid molecule
microrna
mir
recognition sequence
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廖微曦
刘乙齐
曹玉冰
郭亚琨
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Beijing Syngentech Co Ltd
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Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to an oncolytic virus system that specifically kills tumor cells and its application. More specifically, the present invention relates to expression systems, recombinant viruses, recombinant cells, and expression systems, recombinant viruses, and recombinant cells. Use in the preparation of medicines and pharmaceutical compositions.
  • Oncolytic virus refers to a type of virus that has the ability to replicate and package to achieve tumor killing. At present, most studies have modified some of the weaker virulence species that exist in nature to specifically express and package them in tumor cells to achieve oncolysis.
  • the expression of key genes allows oncolytic viruses to replicate in tumor cells in large quantities and express toxic proteins to destroy tumor cells, and/or secrete cytokines at the same time to stimulate the immune system to attack tumor cells.
  • oncolytic viruses cannot replicate in normal body cells without killing effects, so oncolytic viruses have higher anti-tumor effects and lower side effects.
  • oncolytic virus therapy has attracted widespread attention, and related research has made great progress.
  • adenovirus, herpes simplex virus-1 (HSV-1), and Newcastle disease virus have been transformed into oncolytic viruses.
  • oncorine oncolytic adenovirus products (oncorine) have been used in clinical treatments in China, mainly for the treatment of head and neck tumors and sinus cancer. Gendicine and oncorine have similar principles.
  • the E1B-55kD region of human type 5 adenovirus is deleted so that the virus can multiply in cancer cells with p53 gene mutations and kill host cells, resulting in oncolytic therapy.
  • JX-594 from American biotherapy company Jennerex is a modified vaccinia virus.
  • the median life extension time of patients with primary liver cancer after being injected with high doses of the virus can reach 14.1 months, while patients receiving low-dose injections only have 6.7 months Extension of life.
  • the drug is currently in the phase III clinical stage of liver cancer treatment.
  • OncoVex The genetically engineered herpes simplex virus OncoVEX GM-CSF developed by the biotechnology company BioVex was approved by the FDA in October 2015 and became the first oncolytic virus product marketed in the United States and Europe. OncoVex can selectively kill tumor cells while expressing and secreting GM-CSF to initiate the body's immune response to kill the remaining tumor cells and their metastatic sites.
  • the results of a phase II trial of metastatic melanoma published by BioVex in 2009 showed that 26% of 50 patients responded to treatment, and 8 patients recovered completely.
  • the company was acquired by Agmen for US$1 billion in 2011 to advance Phase III clinical trials.
  • Amgen announced the treatment data of OncoVex, which clinically proved that it can successfully shrink tumors in advanced patients.
  • Amgen’s drug is better than similar Other drugs performed even better.
  • oncolytic viruses do have great application prospects in targeted tumor therapy.
  • the current traditional oncolytic virus research and development platforms still have the problems of single regulation, poor targeting, and low platform transformation capabilities.
  • the inventor constructed a closed loop of mutual inhibition in response to multiple input signals. This loop regulates the expression of E1A through switches and then regulates the expression, replication and packaging of adenovirus in cancer cells. In the closed loop, it is unnecessary to remove some virus packaging.
  • the gene reduces the toxicity of the virus to non-target cells, while increasing the packaging capacity of the virus, such as E3, E4, etc., replacing the coat protein of the adenovirus, and changing the virus's targeting of specific cells and tissues.
  • the inventors were consciously surprised to find that in the closed circuit, certain tumor cell-specific promoters have a significantly higher ability to flip some specific tumor cell microRNAs in specific tumor cells. However, some tumor cell-specific promoters have low or no flipping ability for their specific microRNA in some tumor cells. Based on this, the inventors proposed a closed circuit specific to a certain cancer based on the previous research platform.
  • the closed circuit has a high turnover ability in specific cancer cells.
  • the oncolytic virus carrying the closed circuit is effective against specific cancer cells. The killing efficiency is high and effective, and it does not kill normal cells, and the safety is higher.
  • the present invention proposes an expression system.
  • the system includes: a first nucleic acid molecule, the first nucleic acid molecule containing a cell-specific promoter; a second nucleic acid molecule, the second nucleic acid molecule is operable with the first nucleic acid molecule Ground connection, the second nucleic acid molecule encodes a transcription activator; a third nucleic acid molecule, the third nucleic acid molecule contains the first recognition sequence of the transcription activator; a fourth nucleic acid molecule, the fourth nucleic acid molecule and the The third nucleic acid molecule is operably connected, the fourth nucleic acid molecule contains a first promoter and a first regulatory element; a fifth nucleic acid molecule, the fifth nucleic acid molecule is operably connected to the fourth nucleic acid molecule, and the The fifth nucleic acid molecule encodes the first regulatory protein and the target protein, and the target protein includes at least one selected from
  • the expression system may further include at least one of the following additional technical features:
  • the first recognition sequence and the second recognition sequence are independently selected from at least one of UAS, tetO and dCas9 target sequences.
  • the number of repetitions of the UAS or tetO segment can be adjusted as required, for example, 2 ⁇ UAS, 3 ⁇ UAS, 4 ⁇ UAS, or 5 ⁇ UAS, 5 ⁇ tetO, 6 ⁇ tetO, or 7 ⁇ tetO can be selected.
  • the first identification sequence and the second identification sequence are 5 ⁇ UAS.
  • the inventors unexpectedly discovered in experiments that the use of cancer-specific promoter CEA368 and cancer-specific microRNA mir-21 and mir-143-3p regulated switch circuit-controlled oncolytic adenovirus, the use of cancer-specific promoter CEA205 and cancer-specific microRNA mir Oncolytic adenovirus controlled by the switch circuit controlled by -21 and mir-135a-5p, oncolytic adenovirus controlled by the switch circuit controlled by the cancer-specific promoter CEA368 and cancer-specific microRNA mir-21 and mir-135a-5p, use Cancer-specific promoter CXCR4 and cancer-specific microRNA mir-21 and mir-135a-5p regulated switch circuit-controlled oncolytic adenovirus, using cancer-specific promoter Survivin1 and cancer-specific microRNA mir-21 and mir-135a-5p regulation
  • the oncolytic adenovirus controlled by the switch circuit can specifically kill the gastric cancer cell line AGS. Compared with the 4
  • the transcription activator is at least one selected from Gal4VP16, Gal4VP64, Gal4esn, dCas9-VP16, dCas9-VP64, dCas9-VPR, dCas9-VTR and rtTA.
  • the first recognition sequence and the second recognition sequence are independently selected from at least one of 5 ⁇ UAS, 7 ⁇ tetO, and dCas9 target sequences.
  • the first promoter and the second promoter are independently selected from miniCMV and TATA box.
  • the first regulatory protein and the second regulatory protein are independently selected from Lacl, tetR, zinc finger (zinc finger), TALE, KRAB, tetR-KRAB, TALE-KRAB, dCas9-KRAB At least one of miniCas9-KRAB, split dCas9-KRAB.
  • the first regulatory element and the second regulatory element are independently selected from tetO, LacO, zinc finger target site, TALE protein target sequence, and dCas9 target sequence. And at least one of the target sequences of miniCas9.
  • the first regulatory protein is LacI
  • the second regulatory element includes a plurality of repeated LacO sequences, and at least one of the plurality of repeated LacO sequences is set in the second promoter Downstream. After LacI is expressed, it can specifically bind to LacO sequence, thereby inhibiting the function of the second promoter.
  • LacI/LacO suppression system of the embodiment of the present invention experiments show that the system can effectively suppress the expression of genes downstream of the promoter.
  • the second regulatory protein is tetR-KRAB.
  • the first regulatory element includes a plurality of repeated tetO sequences, and at least one of the plurality of repeated tetO sequences is set in the first promoter. The downstream of the child.
  • the tetR-KRAB/tetO suppression system according to the embodiment of the present invention can effectively suppress the expression of genes downstream of the promoter.
  • the viral replication packaging protein and immune effector may exist in the form of a fusion protein.
  • the viral replication packaging protein can effectively ensure the survival and replication of the expression system vector in the host; the expression of immune effector factors can effectively activate the body's immune system, thereby promoting the immune killing of gastric cancer cells and pancreatic cancer cells.
  • the viral replication packaging-related protein includes at least one selected from the group consisting of adenovirus E1 gene, E1A gene, E1B gene, E2 gene, and E4 gene.
  • the immune effector includes an inhibitory sequence selected from the group consisting of an inhibitory sequence against PD-1 gene, an inhibitory sequence against PD-L1 gene, an inhibitory sequence against CTLA4 gene, an inhibitory sequence against Tim-3 gene, GM -At least one of CSF, IL-2, IL-12, IL-15.
  • the aforementioned immune effector may exist in the form of a fusion protein.
  • the target protein and the first regulatory protein are expressed in the form of a fusion protein, and the target protein and the first regulatory protein are connected by a cleavable connecting peptide.
  • the target protein and the first regulatory protein are regulated and expressed under the same promoter, and are cleaved at the connecting peptide after expression.
  • the target protein is separated from the first regulatory protein, and the target protein and the first regulatory protein function independently of each other.
  • the first nucleic acid molecule and the second nucleic acid molecule are loaded on a first expression vector
  • the third nucleic acid molecule, the fourth nucleic acid molecule, the fifth nucleic acid molecule, and the The ninth nucleic acid molecule is loaded on a second expression vector
  • the sixth nucleic acid molecule, the seventh nucleic acid molecule, the eighth nucleic acid molecule and the tenth nucleic acid molecule are loaded on a third expression vector.
  • the first, second and third expression vectors are used as load carriers of the expression system to achieve the specific expression of the target protein in gastric cancer cells and pancreatic cancer cells.
  • the selection of the expression vector is not particularly limited, as long as the expression system can specifically function in gastric cancer cells and pancreatic cancer cells.
  • the first expression vector, the second expression vector and the third expression vector are independently selected from at least one of the following:
  • Plasmids, viruses, stable cell lines and other material carriers such as nanomaterials, liposomes, molecularly coupled carriers, naked DNA, chromosomal carriers, polymers.
  • the virus includes at least one selected from the group consisting of adenovirus, vaccinia virus, herpes virus, and retrovirus.
  • the first expression vector, the second expression vector and the third expression vector are constructed and loaded on the same vector.
  • the connection sequence of the first expression vector, the second expression vector and the third expression vector is not particularly limited, as long as it does not affect the realization of the biological function of the system. According to specific embodiments of the present invention, loading on the same expression vector can effectively solve the problem of extremely low co-transfection efficiency of multiple large fragment vectors.
  • the same vector is an adenovirus vector.
  • adenovirus As a gene therapy vector, adenovirus has a wide main range, low pathogenicity to humans, infects and expresses genes in proliferating and non-proliferating cells, high titer, homology with human genes, no insertional mutagenicity, and can be cultured in suspension The advantages of amplification in liquid and the ability to express multiple genes simultaneously.
  • the cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence is identical to the second The recognition sequence is 5 ⁇ UAS; or the cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence is the same as the first The second recognition sequence is 5 ⁇ UAS; or the cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence is the same as the The second recognition sequence is 5 ⁇ UAS; or the cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence is the same as The second recognition sequence is 5 ⁇ UAS; or the cell-specific promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the first recognition sequence is The second recognition sequence is 5 ⁇ UAS; or
  • a recognition sequence and the second recognition sequence are 4 ⁇ UAS; or the cell-specific promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the The first identification sequence and the second identification sequence are 4 ⁇ UAS.
  • the oncolytic adenovirus controlled by the expression system composed of the above-mentioned elements according to the embodiments of the present invention can specifically kill gastric cancer cells.
  • the switch circuit with 5 ⁇ UAS has a greater killing effect than normal gastric cells with 4 ⁇ UAS switch circuit. small.
  • the cell-specific promoter is hMuc1
  • the first microRNA is mir-199a-3p
  • the second microRNA is mir-21
  • the first recognition sequence is identical to the second
  • the recognition sequence is 4 ⁇ UAS.
  • the oncolytic adenovirus controlled by the expression system composed of the above elements according to the embodiment of the present invention can specifically kill pancreatic cancer cells.
  • the adenovirus vector carries a nucleic acid having a nucleotide sequence shown in any one of SEQ ID NO: 1 to 26.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO:1 is encoded by the cell-specific promoter CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 2 is encoded by the cell-specific promoter CEA205, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 3 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of LacI and tetR-KRAB, and the first and second recognition sequences are 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 4 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 5 is encoded by the cell-specific promoter CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are Lacl, tetR-KRAB, and an expression system (SEQ ID NO: 5) composed of elements whose first and second recognition sequences are 5 ⁇ UAS, respectively.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 6 is encoded by the cell-specific promoter CXCR4, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 7 is encoded by the cell-specific promoter Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 8 is encoded by the cell-specific promoter Survivin1, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 9 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 10 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 11 is encoded by the cell-specific promoter CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 12 is encoded by the cell-specific promoter CEA205, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of TALE protein, and the first and second recognition sequences are 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 13 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 14 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of TALE protein, and the first and second recognition sequences are 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 15 is encoded by the cell-specific promoter CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 16 is encoded by the cell-specific promoter CXCR4, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of TALE protein, and the first and second recognition sequences are 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 17 is encoded by the cell-specific promoter Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 18 is encoded by the cell-specific promoter Survivin1, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of TALE protein, and the first and second recognition sequences are 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 19 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 20 is encoded by the cell-specific promoter CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of TALE protein, and the first and second recognition sequences are 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 21 is encoded by the cell-specific promoter hMUC1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 22 is encoded by the cell-specific promoter hMUC1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of Lacl, tetR-KRAB, and the first and second recognition sequences of 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 23 is encoded by the cell-specific promoter hMUC1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of TALE protein, and the first and second recognition sequences are 4 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 24 is encoded by the cell-specific promoter hMUC1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, the transcription activator is Gal4VP16, and the first regulation
  • the protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 25 is encoded by the cell-specific promoter hMUC1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, the transcription activator is segmented dCas9, and the first microRNA is mir-199a-3p.
  • the regulatory protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the nucleic acid with the nucleotide sequence described in SEQ ID NO: 26 is encoded by the cell-specific promoter hMUC1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, the transcription activator is segmented dCas9, the first The regulatory protein and the second regulatory protein are respectively an expression system composed of the TALE protein and the first and second recognition sequences of 5 ⁇ UAS elements.
  • the adenovirus is obtained in the following manner: the adenovirus vector removes the E1 gene and part of the E3 gene related to adenovirus replication packaging, and the E1A gene is constructed by a step-by-step Golden Gate method Into the gene circuit, the gene circuit is finally inserted into the adenovirus vector through Gateway or Gibson.
  • the above-mentioned method of obtaining adenovirus realizes the rapid transformation of complex and large-segment oncolytic adenovirus vector.
  • the specific construction method can refer to 201780002478.X.
  • the adenovirus vector is an adenovirus of subtypes B and C with E1 gene and part of E3 gene removed.
  • the adenovirus vector is an adenovirus of type 5, 11, 12, 34 or 35 with the E1 gene and part of the E3 gene removed.
  • the nucleotide sequence shown in any one of SEQ ID NO: 1 to 26 is inserted into the type 5 adenovirus vector at the E1 gene region, E3 gene region or E4 gene region.
  • the map of the type 5 adenovirus vector with the E1 gene and part of the E3 gene removed can be seen in Figure 13.
  • the sequence is shown in SEQ ID NO: 27, and the insertion site can be selected at the 459th base after the 458th base of the sequence. Bases before.
  • the present invention proposes a recombinant virus.
  • the recombinant virus includes: a first nucleic acid molecule, the first nucleic acid molecule containing a tumor cell-specific promoter; a second nucleic acid molecule, the second nucleic acid molecule and the first nucleic acid molecule Operably linked, the second nucleic acid molecule encodes a transcription activator, the transcription activator is Gal4VP16; a third nucleic acid molecule, the third nucleic acid molecule contains the first recognition sequence of the transcription activator; a fourth nucleic acid The fourth nucleic acid molecule is operably linked to the third nucleic acid molecule, the fourth nucleic acid molecule contains a first promoter and a first regulatory element, the first promoter is miniCMV, and the first The control element includes a plurality of repeated tetO sequences, at least one of the plurality of repeated tetO sequences is
  • the first identification sequence and the second identification sequence are 4 ⁇ UAS.
  • the first regulatory protein LacI and the target protein are specifically expressed in gastric cancer and pancreatic cancer cells under the common regulation of a tumor cell-specific promoter, the ninth nucleic acid molecule, and the tenth nucleic acid molecule.
  • the second regulatory protein tetR-KRAB is specifically not expressed or underexpressed in gastric cancer and pancreatic cancer cells, and the suppression mechanism of the first promoter miniCMV mediated by tetR-KRAB is lifted.
  • the first regulatory protein LacI and the target protein are in the first A promoter miniCMV is effectively expressed under the start-up regulation, LacI-mediated suppression mechanism effectively inhibits the function of the second promoter miniCMV, and the expression of tetR-KRAB is further suppressed.
  • LacI-mediated suppression mechanism effectively inhibits the function of the second promoter miniCMV
  • the expression of tetR-KRAB is further suppressed.
  • more specific expression of the protein such as the target protein or LacI
  • no expression such as tetR-KRAB
  • the target protein in the expression efficiency and specificity are high.
  • the recombinant virus according to the embodiment of the present invention can achieve specific, efficient and safe killing of gastric cancer and pancreatic cancer cells.
  • the aforementioned recombinant virus may further include at least one of the following additional technical features:
  • the recombinant virus is at least one selected from retrovirus, adenovirus, herpes virus, and vaccinia virus.
  • the recombinant virus is an adenovirus.
  • adenovirus as a gene therapy vector has a wide host range, low pathogenicity to humans, infects and expresses genes in proliferating and non-proliferating cells, high titer, homology with human genes, and no insertional mutagenicity , It can be amplified in suspension culture and can express multiple genes at the same time.
  • the immune effector includes an inhibitory sequence selected from the group consisting of an inhibitory sequence against PD-1 gene, an inhibitory sequence against PD-L1 gene, an inhibitory sequence against CTLA4 gene, an inhibitory sequence against Tim-3 gene, IL -2. At least one of IL-15, IL-12, GM-CSF.
  • the immune effector may be in the form of a fusion protein.
  • the present invention proposes a recombinant cell.
  • the recombinant cell contains the aforementioned expression system.
  • the recombinant cells according to the embodiments of the present invention can effectively activate the human body's systemic immune response, specifically attack gastric cancer and pancreatic cancer cells, with high safety and strong specificity.
  • the aforementioned recombinant cell may further include at least one of the following additional technical features:
  • At least a part of the expression system is integrated into the genome of the recombinant cell.
  • the expression system replicates with the replication of the recombinant cell genome, and the expression system regulates the expression of the target protein continuously and effectively.
  • the present invention proposes the use of the aforementioned expression system, the aforementioned recombinant virus, and the aforementioned recombinant cell in the preparation of medicines for the treatment of gastric cancer or pancreatic cancer.
  • the present invention proposes a pharmaceutical composition.
  • the pharmaceutical composition comprises the aforementioned recombinant virus or the aforementioned recombinant cell.
  • the pharmaceutical composition according to the embodiment of the present invention has a significant therapeutic effect on gastric cancer or pancreatic cancer.
  • the aforementioned pharmaceutical composition may further include pharmaceutically acceptable excipients.
  • the pharmaceutical composition further includes other drugs for treating gastric cancer.
  • the other drugs for treating gastric cancer include at least one selected from Pembrolizumab, Ogivri, Everolimus, Lanreotide, Ramucirumab, Apatinib, and Trastuzumab.
  • the pharmaceutical composition further includes other drugs for treating pancreatic cancer.
  • the other drugs for treating pancreatic cancer include at least one selected from Lanreotide, Abraxane, Olaparib, Afinitor, Erlotinib, Everolimus, 5-FU, Gemzar, Sunitinib, Onivyde, and Gemzar.
  • the pharmaceutical composition of the present invention can be administered when treating or preventing gastric cancer and pancreatic cancer.
  • administration refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner.
  • the pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the intended tissue.
  • Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the present invention is not limited to these exemplified modes of administration.
  • the active ingredient of the oral administration composition should be coated or formulated to prevent its degradation in the stomach.
  • the composition of the present invention may be administered as an injection formulation.
  • the pharmaceutical composition of the present invention can be administered using a specific device that delivers the active ingredient to the target cell.
  • the administration frequency and dosage of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the route of administration, the patient’s age, sex, weight, and the severity of the disease as well as the active ingredient Type of drug.
  • the daily dose can be divided into 1 dose, 2 doses or multiple doses in a suitable form, so as to be administered once, twice or multiple times in the entire time period, as long as the therapeutically effective amount is reached .
  • terapéuticaally effective amount refers to an amount of a compound that is sufficient to significantly improve certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
  • drugs or compounds that reduce, prevent, delay, inhibit, or block any symptoms of the disease or disorder should be therapeutically effective.
  • a therapeutically effective amount of a drug or compound does not need to cure the disease or condition, but will provide treatment for the disease or condition so that the onset of the disease or condition of the individual is delayed, prevented, or prevented, or the symptoms of the disease or condition are alleviated, or the disease or condition The duration of the illness is changed, or, for example, the disease or illness becomes less serious, or recovery is accelerated.
  • treatment is used to refer to obtaining the desired pharmacological and/or physiological effect.
  • the effect may be preventive in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease.
  • Treatment covers the treatment of diseases (mainly gastric cancer or pancreatic cancer) in mammals, especially humans, including: (a) prevention of diseases in individuals who are prone to disease but have not yet been diagnosed with the disease (for example, prevention of gastric cancer or pancreatic cancer) Cancer) or the occurrence of a disorder; (b) inhibiting the disease, such as blocking the development of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease.
  • Treatment encompasses any medication that administers a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit the individual's disease, including but not limited to administering the drug containing the herein described to an individual in need.
  • the pharmaceutical composition of the present invention may be used in combination with conventional treatment methods and/or therapies, or may be used separately from conventional treatment methods and/or therapies.
  • the drugs of the present invention are administered in combination therapy with other drugs, they can be administered to the individual sequentially or simultaneously.
  • the pharmaceutical composition of the present invention may comprise a combination of the recombinant virus of the present invention or a pharmaceutically acceptable excipient and other therapeutic or preventive drugs known in the art.
  • the fusion protein described in this application refers to a protein co-transcribed under the control of the same promoter, including a fusion protein without a link between the proteins, or with other linking peptides (such as GGGS or 2A sequence). ) Linked fusion protein.
  • the "flipping ability" of the closed loop or expression system described in this application refers to the difference in output levels when controlled by the input signals of the first microRNA and the second microRNA, which is specifically embodied in the target protein (including selected viruses Copy at least one of the packaging protein and immune effector) the difference in expression level.
  • severe ⁇ sequence refers to the sequential connection of several repeated sequences with no intervening bases between the repeated sequences.
  • 5 ⁇ UAS refers to a sequence composed of five repeated UAS successfully connected.
  • 6 ⁇ tetO refers to a sequence composed of 6 repeated tetOs connected sequentially.
  • Figure 1 is a schematic diagram of the construction and testing process of an oncolytic adenovirus targeting specific tumors according to an embodiment of the present invention
  • Figure 2 shows the expression test of different cancer-specific promoters in gastric cancer and normal gastric cell lines according to an embodiment of the present invention
  • Figure 3 is a test of flipping switch circuits of different cancer-specific promoters in gastric cancer and normal gastric cell lines according to an embodiment of the present invention
  • Figure 4 is a test of the inhibition efficiency of different microRNA target sites in gastric cancer and normal gastric cell lines according to an embodiment of the present invention
  • 5A to 5J are the killing ability tests of different adenoviruses in gastric cancer and normal cell lines according to embodiments of the present invention.
  • Fig. 6 is an expression test of different cancer-specific promoters in breast cancer and normal breast cell lines according to an embodiment of the present invention
  • Figure 7 is a test of flipping switch circuits of different cancer-specific promoters in breast cancer and normal breast cell lines according to an embodiment of the present invention.
  • Fig. 8 shows the inhibition efficiency test of different microRNA target sites in breast cancer and normal breast cell lines according to an embodiment of the present invention.
  • Figures 9A-9D show the killing ability tests of different adenoviruses in breast cancer and normal cell lines according to embodiments of the present invention.
  • Figure 10 shows the expression test of different cancer-specific promoters in pancreatic cancer and normal pancreatic cell lines
  • Figure 11 is a test of flipping switch circuits of different cancer-specific promoters in pancreatic cancer and normal pancreatic cell lines according to an embodiment of the present invention
  • Figure 12 shows the killing ability test of different adenoviruses in pancreatic cancer and normal cell lines according to an embodiment of the present invention.
  • Figure 13 is a map of a type 5 adenovirus vector according to an embodiment of the present invention.
  • the involved method and test procedure for constructing an oncolytic adenovirus targeting specific tumors can be referred to the schematic diagram, Fig. 1; the virus codes involved are shown in Table 1.
  • Example 1 Construction and functional verification of oncolytic adenovirus targeting gastric cancer
  • CEA205-Gal4VP16 plasmid, UAS-EYFP plasmid, and hEF1a-EBFP2 plasmid were co-transfected into gastric cancer cell line AGS and gastric normal cell line GES1 (100ng each plasmid was transfected in each well), and flow cytometry was performed 48 hours after transfection Analyze and detect the fluorescence intensity of EYFP and EBFP2.
  • Normalized reporter gene expression level (fluorescence intensity of experimental group EYFP/experimental group EBFP2 fluorescence intensity)/(control group EYFP fluorescence intensity/control group EBFP2 fluorescence intensity).
  • CEA368-Gal4VP16 plasmid instead of the CEA205-Gal4VP16 plasmid, and perform the above steps.
  • Use the cerbB2-Gal4VP16 plasmid instead of the CEA205-Gal4VP16 plasmid, and perform the above steps.
  • Use the COX2-Gal4VP16 plasmid instead of the CEA205-Gal4VP16 plasmid, and perform the above steps.
  • Use CXCR4-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, and perform the above steps.
  • Use hMUC1-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, perform the above steps.
  • the normalized reporter gene expression level is shown in Figure 2.
  • the expression levels of all cancer-specific promoters in the normal gastric cell line GES1 were very low, while in the gastric cancer cell line AGS, the expression levels of CEA205, CEA368, CXCR4 and Survivin1 were higher.
  • the results showed that cancer-specific promoters can specifically initiate the expression of Gal4VP16 in gastric cancer cell lines and activate reporter genes downstream of UAS.
  • CEA205, CEA368, CXCR4 and Survivin1 have good expression intensity and specificity.
  • CEA368-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, 4 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4 After ⁇ UAS-LacO-TetRKrab-FF5 plasmid replaces 5 ⁇ UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, and perform the above steps; use U6-shRNA- FF5-CMV-iRFP plasmid replaces pDT7004 plasmid, and the above steps are performed.
  • CAG-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, 4 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4 After ⁇ UAS-LacO-TetRKrab-FF5 plasmid replaces 5 ⁇ UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, and perform the above steps; use U6-shRNA- FF5-CMV-iRFP plasmid replaces pDT7004 plasmid, and the above steps are performed.
  • the expression level of the reporter gene is shown in Figure 3. Consistent with the results of experiment 1, all the tested promoters expressed very low expression in the normal gastric cell line GES1, while in the gastric cancer cell line AGS, the switch circuit could be reversed.
  • the expression levels of CEA368 and Survivin1 in the gastric cancer cell line AGS were slightly higher than those of CEA205 and CXCR4, while the expression intensity of 5 ⁇ UAS switch circuit was slightly higher than that of 4 ⁇ UAS switch circuit.
  • the results show that all the tested promoters can specifically initiate 4 ⁇ UAS or 5 ⁇ UAS switch circuit inversion in the gastric cancer cell line AGS.
  • the CMV-EYFP-T143 3p x4 plasmid, CMV-EBFP2 plasmid, and pDT7004 plasmid were co-transfected into gastric cancer cell line AGS and gastric normal cell line GES1 (100ng each plasmid was transfected in each well), and flow cytometry was performed 48 hours after transfection Technical analysis to detect the fluorescence intensity of EYFP and EBFP2.
  • Normalized reporter gene expression level (experimental group EBFP2 fluorescence intensity/experimental group EYFP fluorescence intensity)/(control group EBFP2 fluorescence intensity/control group EYFP fluorescence intensity).
  • the normalized reporter gene expression level is shown in Figure 4.
  • mir-21 can specifically inhibit the expression of reporter genes in gastric cancer cell line AGS through the target site, and has a high inhibitory strength.
  • mir-143-3p and mir-135a-5p can specifically inhibit the expression of reporter genes in the normal gastric cell line GES1 through the target site, and have a certain inhibitory strength.
  • the results showed that cancer-specific high- or low-expressed microRNAs can specifically inhibit reporter gene expression in gastric cancer cell line AGS or gastric normal cell line GES1 through target sites.
  • Cancer-specific high-expressing microRNA mir-21, cancer-specific low-expressing microRNA mir-143-3p and mir-135a-5p have good inhibitory strength.
  • Inoculate 1e4 cells per well in a 96-well plate Inoculate 1e4 cells per well in a 96-well plate.
  • A1 virus with a multiplicity of infection of 1, 10, 20, 50, 100, 200, 500 was added 24 hours after inoculation, and a blank control without virus was set.
  • the MTS method was used to detect the cell survival rate after 6 days of virus infection.
  • Cell survival rate MTS detection value of the experimental group / MTS detection value of the control group.
  • Use A2 virus instead of A1 virus to perform the above steps Use A3 virus instead of A1 virus to perform the above steps.
  • Use A4 virus instead of A1 virus to perform the above steps.
  • Use A5 virus instead of A1 virus to perform the above steps.
  • Use A6 virus instead of A1 virus to perform the above steps.
  • Use A7 virus instead of A1 virus to perform the above steps.
  • Use A8 virus instead of A1 virus to perform the above steps.
  • the cell survival rate is shown in Figures 5A to 5J. Consistent with the results of experiments 1, 2, and 3, the oncolytic gland controlled by the cancer-specific promoter CEA205/CEA368/CXCR4/Survivin1 and the cancer-specific microRNA mir-21 and mir-135a-5p/mir-143-3p
  • the virus can specifically kill the gastric cancer cell line AGS, but has no obvious killing effect on the normal gastric cell line GES1 and the normal liver cell line Chang and L02.
  • the oncolytic adenovirus A1 controlled by the 5 ⁇ UAS switch circuit showed obvious killing specificity when the multiplicity of infection was about 10-20, while the killing effects of A2, A4, A5 and A1 were similar.
  • the killing effect of oncolytic adenovirus A10 and A1 controlled by 4 ⁇ UAS switch circuit is similar.
  • the results show that the oncolytic adenovirus controlled by the switch circuit controlled by the cancer-specific promoter CEA205/CEA368/Survivin1 and the cancer-specific microRNA mir-21 and mir-135a-5p/mir-143-3p can specifically kill gastric cancer cell lines AGS, 5 ⁇ UAS switch circuit has better effect than 4 ⁇ UAS switch circuit.
  • Example 2 the expression intensity and specificity of cancer-specific promoters CEA205, CEA368, CXCR4, hMUC1 and Survivin1 in breast cancer cell lines MCF7, MDA-MB-231 and normal breast cell line MCF10A were determined.
  • CEA205-Gal4VP16 plasmid, UAS-EYFP plasmid, CMV-EBFP2 plasmid were co-transfected into breast cancer cell lines MCF7, MDA-MB-231 and normal breast cell line MCF10A (100ng each plasmid was transfected in each well), transfected 48 After hours, flow cytometry analysis was performed to detect the fluorescence intensity of EYFP and EBFP2.
  • Normalized reporter gene expression level (fluorescence intensity of experimental group EYFP/experimental group EBFP2 fluorescence intensity)/(control group EYFP fluorescence intensity/control group EBFP2 fluorescence intensity).
  • CEA368-Gal4VP16 plasmid instead of the CEA205-Gal4VP16 plasmid, and perform the above steps.
  • Use CXCR4-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, and perform the above steps.
  • Use hMUC1-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, perform the above steps.
  • Use Survivin1-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid to perform the above steps.
  • Use CAG-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid to perform the above steps.
  • the normalized reporter gene expression level is shown in Figure 6.
  • CEA205 and CXCR4 are more highly expressed in breast cancer cell lines MCF7 and MDA-MB-231, but lower in normal breast cell line MCF10A.
  • the results show that cancer-specific promoters can specifically initiate the expression of Gal4VP16 in breast cancer cell lines and activate reporter genes downstream of UAS.
  • CEA205 and CXCR4 have good expression intensity and specificity.
  • CEA368-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, 4 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4 After ⁇ UAS-LacO-TetRKrab-FF5 plasmid replaces 5 ⁇ UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, and perform the above steps; use U6-shRNA- FF5-CMV-iRFP plasmid replaces pDT7004 plasmid, and the above steps are performed.
  • CAG-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, 4 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4 After ⁇ UAS-LacO-TetRKrab-FF5 plasmid replaces 5 ⁇ UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, and perform the above steps; use U6-shRNA- FF5-CMV-iRFP plasmid replaces pDT7004 plasmid, and the above steps are performed.
  • the expression level of the reporter gene is shown in Figure 7.
  • the expression level of the promoter flipped 5 ⁇ UAS switch circuit was low, while the flipped 4 ⁇ UAS switch circuit had a certain expression.
  • the startup tested in the breast cancer cell line MCF7 failed to show better flipping ability for the 5 ⁇ UAS and 4 ⁇ UAS switch circuits.
  • the results show that the tested promoter does not have a strong ability to flip the switch circuit in the breast cancer cell line MCF7.
  • the CMV-EYFP-T205 5p x4 plasmid, CMV-EBFP2 plasmid, and pDT7004 plasmid were co-transfected into breast cancer cell lines MCF7, MDA-MB-231 and normal breast cell line MCF10A (100ng each plasmid was transfected in each well), and transfected Flow cytometry analysis was performed 48 hours later to detect the fluorescence intensity of EYFP and EBFP2.
  • CMV-EYFP plasmid instead of CMV-EYFP-T205 5p x4 plasmid.
  • Normalized reporter gene expression level (experimental group EBFP2 fluorescence intensity/experimental group EYFP fluorescence intensity)/(control group EBFP2 fluorescence intensity/control group EYFP fluorescence intensity).
  • mir-205-5p can specifically inhibit reporter gene expression in normal breast cell line MCF10A through the target site, and has a high inhibitory strength, which is consistent with the survey results.
  • mir-141-3p has a lower inhibitory strength in the breast cancer cell line MDA-MB-231, it has a certain inhibitory strength in MCF7.
  • mir-21 has a high inhibitory strength in breast cancer cell lines MCF7 and MDA-MB-231, it has a high inhibitory strength in normal breast cell line MCF10A.
  • the results show that cancer-specific high- or low-expressed microRNAs can inhibit reporter gene expression in breast cancer cell lines MCF7, MDA-MB-231 and normal breast cell lines MCF10A through target sites, but the specificity is not good enough.
  • an oncolytic adenovirus controlled by the 5 ⁇ UAS switch circuit controlled by the CEA205/Survivin1 promoter, mir-21 and mir-141-3p was constructed, and tested against breast cancer cell lines MCF7, MDA -The killing ability of MB-231, normal breast cell line MCF10A, normal liver cell line Chang and L02.
  • the cell survival rate is shown in Figures 9A-9D.
  • the four adenoviruses failed to effectively kill the breast cancer cell lines MCF7 and MDA-MB-231, but had a certain killing effect on the normal breast cell line MCF10A.
  • the results showed that the oncolytic adenovirus controlled by the switch circuit controlled by the cancer-specific promoter CEA205/Survivin1 and cancer-specific microRNA mir-21 and mir-141-3p failed to specifically kill the breast cancer cell lines MCF7 and MDA-MB- 231.
  • Example 2 the expression intensity and specificity of the cancer-specific promoter hMUC1 in the pancreatic cancer cell line PANC1 and the pancreatic normal cell line HTERT-HPNE were determined.
  • CAG-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid to perform the above steps.
  • hMUC1 is highly expressed in the pancreatic cancer cell line PANC1 and low in the normal pancreatic cell line HTERT-HPNE. The results show that the cancer-specific promoter hMUC1 can specifically initiate the expression of Gal4VP16 in breast cancer cell lines and activate the reporter gene downstream of UAS.
  • CAG-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid, 4 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5 ⁇ UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4 After ⁇ UAS-LacO-TetRKrab-FF5 plasmid replaces 5 ⁇ UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, and perform the above steps; use U6-shRNA- FF5-CMV-iRFP plasmid replaces pDT7004 plasmid, and the above steps are performed.
  • hMUC1 can flip the 5 ⁇ UAS and 4 ⁇ UAS switch circuits, but it is also expressed in the normal pancreatic cell line HTERT-HPNE.
  • the results show that hMUC1 has a certain ability to flip the switch circuit in the pancreatic cancer cell line PANC1, but it has a certain leakage in the normal pancreatic cell line HTERT-HPNE.
  • Inoculate 1e4 cells per well in a 96-well plate Inoculate 1e4 cells per well in a 96-well plate.
  • C1 virus with a multiplicity of infection of 0.1, 1, 5, 10, 20, 50, 100 was added 24 hours after inoculation, and a blank control without virus was set.
  • the MTS method was used to detect the cell survival rate after 6 days of virus infection.
  • Cell survival rate MTS detection value of the experimental group / MTS detection value of the control group.
  • the cell survival rate is shown in Figure 12. Both adenoviruses can effectively and specifically kill the pancreatic cancer cell line PANC1. C1 showed obvious killing specificity when the multiplicity of infection was about 20-50, while the killing effect of C2 was slightly weaker than pAD043. The results show that the oncolytic adenovirus controlled by the switch circuit controlled by the cancer-specific promoter hMUC1 and cancer-specific microRNA mir-21 and mir-199a-3p can specifically kill the pancreatic cancer cell line PANC1

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Abstract

L'invention concerne un système d'expression, un virus recombinant contenant ledit système d'expression, et une cellule recombinante contenant ledit système d'expression. L'invention concerne également l'utilisation du système d'expression, du virus recombinant ou de la cellule recombinante dans la préparation d'un médicament pour le traitement ou la prévention du cancer gastrique ou du cancer du pancréas.
PCT/CN2020/083823 2019-05-31 2020-04-08 Système de virus oncolytique pour tuer spécifiquement des cellules tumorales, et son application Ceased WO2020238427A1 (fr)

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