WO2020238427A1 - Oncolytic virus system for specifically killing tumor cells, and application thereof - Google Patents

Oncolytic virus system for specifically killing tumor cells, and application thereof 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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北京合生基因科技有限公司
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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

Abstract

Provided are an expression system, a recombinant virus containing said expression system, and a recombinant cell containing said expression system. Also provided are the use of the expression system, the recombinant virus, or the recombinant cell in the preparation of a drug for treating or preventing gastric cancer or pancreatic cancer.

Description

特异杀伤肿瘤细胞的溶瘤病毒系统及其应用Oncolytic virus system specifically killing tumor cells and its application 技术领域Technical field
本发明涉及生物医药领域，具体地，本发明涉及特异杀伤肿瘤细胞的溶瘤病毒系统及其应用，更具体地，本发明涉及表达系统、重组病毒、重组细胞以及表达系统、重组病毒、重组细胞在制备药物中的用途以及药物组合物。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.
背景技术Background technique
溶瘤病毒是指一类具有复制与包装能力实现肿瘤杀伤作用的病毒。目前，多数研究通过改造一些自然界存在的致病力较弱的毒种，使其特异的在肿瘤细胞中表达包装继而实现溶瘤作用。利用溶瘤病毒识别肿瘤细胞的原理主要有两种：第一，利用靶细胞中抑癌基因的失活或缺陷从而选择性地感染肿瘤细胞；第二，选择利用肿瘤特异性的启动子调控病毒关键基因的表达使溶瘤病毒在肿瘤细胞内大量复制并表达毒性蛋白摧毁肿瘤细胞，并/或同时分泌细胞因子刺激自身免疫系统攻击肿瘤细胞。相应的溶瘤病毒无法在正常机体细胞内复制而不具有杀伤作用，因此溶瘤病毒具有更高的抗肿瘤效应和更低的副作用。近几十年来，溶瘤病毒治疗引起了广泛关注，相关研究取得了巨大进展。目前腺病毒(adenovirus)、单纯疱疹病毒-1(herpes simplex virus-1，HSV-1)、新城疫病毒等相继被改造成溶瘤病毒。2006年，溶瘤腺病毒产品(oncorine)在中国已经用于临床治疗，主要用于治疗头颈部肿瘤、鼻窦癌等。Gendicine和oncorine的原理类似，将人5型腺病毒E1B-55kD区删除，使该病毒可在p53基因突变的癌细胞中繁殖并杀伤宿主细胞，产生溶瘤治疗作用。但是，临床数据显示这两种基于单一p53基因突变的溶瘤腺病毒的治疗效果并不十分理想。美国生物治疗公司Jennerex的JX-594是一种经过修改的牛痘病毒。在2013年完成的二期临床试验中，发现原发性肝癌患者在注射了高剂量的病毒后，其生命延长时间的中间值可达到14.1个月，而接受低剂量注射的患者只有6.7个月的生命延长期。目前该药物已经处于肝癌治疗的三期临床阶段。生物技术公司BioVex研发的基因工程化的单纯疱疹病毒OncoVEX GM-CSF已于2015年10月通过了FDA的批准成为美国和欧洲首个上市的溶瘤病毒产品。OncoVex可选择性地杀灭肿瘤细胞同时表达分泌GM-CSF启动机体产生系统的免疫反应杀伤剩余肿瘤细胞及其转移位点。2009年BioVex公布的一项转移性黑色素瘤II期试验的结果显示50名患者中有26％对治疗有反应，有8名患者完全恢复健康。该公司于2011年被Agmen以10亿美元的价格收购，用于推进三期临床试验。2013年3月，安进(Amgen)公布了OncoVex的治疗数据，临床证明它能成功地让晚期患者的肿瘤缩小，并在超过400名试验患者的III 期研究中，Amgen的该药物要比同类其他药物表现得更加出色。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. There are two main principles for using oncolytic viruses to identify tumor cells: First, use the inactivation or defect of tumor suppressor genes in target cells to selectively infect tumor cells; second, choose to use tumor-specific promoters to regulate viruses 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. Corresponding oncolytic viruses cannot replicate in normal body cells without killing effects, so oncolytic viruses have higher anti-tumor effects and lower side effects. In recent decades, oncolytic virus therapy has attracted widespread attention, and related research has made great progress. At present, adenovirus, herpes simplex virus-1 (HSV-1), and Newcastle disease virus have been transformed into oncolytic viruses. In 2006, 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. However, clinical data shows that the therapeutic effects of these two oncolytic adenoviruses based on a single p53 gene mutation are not very satisfactory. JX-594 from American biotherapy company Jennerex is a modified vaccinia virus. In the Phase II clinical trial completed in 2013, it was found that 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. 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. In March 2013, Amgen announced the treatment data of OncoVex, which clinically proved that it can successfully shrink tumors in advanced patients. In a phase III study of more than 400 trial patients, Amgen’s drug is better than similar Other drugs performed even better.
综上所述，溶瘤病毒确实在肿瘤的靶向治疗方面具有巨大的应用前景。但目前传统的溶瘤病毒研发平台仍然存在调控单一，靶向性差，平台转化能力低的问题。发明人前期构建了一个响应多输入信号的互抑制闭合回路，该回路通过开关调控E1A的表达进而调控腺病毒在癌细胞中的表达、复制和包装，在闭合回路中，去除一些病毒包装非必要的基因减少病毒对非靶标细胞的毒性，同时提高病毒的包装容量，如E3，E4等，替换腺病毒的外壳蛋白，改变病毒对特定细胞和组织的靶向性。In summary, oncolytic viruses do have great application prospects in targeted tumor therapy. However, the current traditional oncolytic virus research and development platforms still have the problems of single regulation, poor targeting, and low platform transformation capabilities. In the early stage, 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.
但基于该闭合回路仍存在较大的改进空间，发明人在前期工作的基础上，对该闭合回路进行了更深入和全面的研究和改进，以使该闭合回路应用于溶瘤病毒后，溶瘤病毒对疾病的治疗更加安全和有效。However, there is still a lot of room for improvement based on the closed circuit. Based on the previous work, the inventor has carried out more in-depth and comprehensive research and improvement on the closed circuit, so that the closed circuit can be applied to oncolytic viruses. Oncoviruses are safer and more effective in treating diseases.
发明内容Summary of the invention
本申请是基于发明人对以下事实和问题的发现和认识作出的：This application is based on the inventor's discovery and understanding of the following facts and problems:
发明人在对前期开发的闭合回路进一步改进的过程中，惊喜地发现，闭合回路中，某些肿瘤细胞特异性启动子在特定肿瘤细胞中对某些特异性肿瘤细胞microRNA的翻转能力显著高，而某些肿瘤细胞特异性启动子在某些肿瘤细胞中对其特异性microRNA的翻转能力却不高或没有翻转能力。基于此，发明人在前期研究平台的基础上，提出了特异性针对某种癌症的闭合回路，该闭合回路在特异癌症细胞中的翻转能力高，承载该闭合回路的溶瘤病毒对特异癌症细胞的杀伤效率高而有效，且对正常细胞没有杀伤，安全性也更高。In the process of further improving the closed circuit developed in the previous stage, the inventors were pleasantly 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.
在本发明的第一方面，本发明提出了一种表达系统。根据本发明的实施例，所述系统包括：第一核酸分子，所述第一核酸分子含有细胞特异性启动子；第二核酸分子，所述第二核酸分子与所述第一核酸分子可操作地连接，所述第二核酸分子编码转录激活因子；第三核酸分子，所述第三核酸分子含有所述转录激活因子的第一识别序列；第四核酸分子，所述第四核酸分子与所述第三核酸分子可操作地连接，所述第四核酸分子含有第一启动子和第一调控元件；第五核酸分子，所述第五核酸分子与第四核酸分子可操作地连接，所述第五核酸分子编码第一调控蛋白和目的蛋白，并且所述目的蛋白包括选自病毒复制包装蛋白、免疫效应因子的至少之一；第六核酸分子，所述第六核酸分子含有所述转录激活因子的第二识别序列；第七核酸分子，所述第七核酸分子与所述第六核酸分子可操作地连接，所述第七核酸分子含有第二启动子和第二调控元件；第八核酸分子，所述第八核酸分子与第七核酸分子可操作地连接，并且所述第八核酸分子编码第二调控蛋白；第九核酸分子，所述第九核酸分子与所述第五核酸分子可操作地连接，所述第九核酸分子被配置为条件性抑制所述第一调控蛋白的表达；以及第十核酸分子，所述第十核酸分子与所述第八核酸分子可 操作地连接，所述第十核酸分子被配置为条件性抑制所述第二调控蛋白的表达，其中，所述第一调控元件适于通过结合所述第二调控蛋白抑制所述第一启动子的功能，所述第二调控元件适于通过结合所述第一调控蛋白抑制所述第二启动子的功能；所述第九核酸分子与所述第十核酸分子分别独立地借助RNA干扰抑制所述第一调控蛋白或所述第二调控蛋白的表达；所述第九核酸分子含有被第一microRNA特异性识别的核酸序列，所述第十核酸分子含有被第二microRNA特异性识别的核酸序列，所述第一microRNA为正常细胞特异性microRNA，所述第二microRNA为异常细胞特异性microRNA；所述细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或所述细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或所述细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或所述细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或所述细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21；或所述细胞特异性启动子为hMuc1，所述第一microRNA为mir-199a-3p所述第二microRNA为mir-21。根据本发明的实施例的表达系统在胃癌、胰腺癌细胞中的翻转能力高，具有该表达系统的表达载体，如腺病毒载体等，对胃癌、胰腺癌细胞的特异性杀伤效率高。In the first aspect of the present invention, the present invention proposes an expression system. According to an embodiment of the present invention, 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 a viral replication packaging protein and an immune effector; a sixth nucleic acid molecule, the sixth nucleic acid molecule containing the transcription activation The second recognition sequence of the factor; the seventh nucleic acid molecule, the seventh nucleic acid molecule is operably linked to the sixth nucleic acid molecule, the seventh nucleic acid molecule contains a second promoter and a second regulatory element; the eighth nucleic acid Molecule, the eighth nucleic acid molecule and the seventh nucleic acid molecule are operably linked, and the eighth nucleic acid molecule encodes a second regulatory protein; the ninth nucleic acid molecule, the ninth nucleic acid molecule and the fifth nucleic acid molecule can be Operably connected, the ninth nucleic acid molecule is configured to conditionally inhibit the expression of the first regulatory protein; and a tenth nucleic acid molecule, the tenth nucleic acid molecule is operably connected to the eighth nucleic acid molecule, and The tenth nucleic acid molecule is configured to conditionally inhibit the expression of the second regulatory protein, wherein the first regulatory element is adapted to inhibit the function of the first promoter by binding to the second regulatory protein, and The second regulatory element is suitable for inhibiting the function of the second promoter by binding to the first regulatory protein; the ninth nucleic acid molecule and the tenth nucleic acid molecule independently inhibit the first regulatory protein by means of RNA interference Or the expression of the second regulatory protein; the ninth nucleic acid molecule contains a nucleic acid sequence specifically recognized by the first microRNA, the tenth nucleic acid molecule contains a nucleic acid sequence specifically recognized by the second microRNA, and the first The microRNA is a normal cell-specific microRNA, the second microRNA is an abnormal cell-specific microRNA; the cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, and the second microRNA is mir- 21; or the cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, and the second microRNA is mir-21; or the cell-specific promoter is CXCR4, the first The microRNA is mir-135a-5p, the second microRNA is mir-21; or the cell-specific promoter is Survivin1, and the first microRNA is mir-135a -5p, the second microRNA is mir-21; or the cell-specific promoter is CEA368, the first microRNA is mir-143-3p, and the second microRNA is mir-21; or the cell The specific promoter is hMuc1, the first microRNA is mir-199a-3p, and the second microRNA is mir-21. The expression system according to the embodiment of the present invention has high turnover ability in gastric cancer and pancreatic cancer cells, and expression vectors with the expression system, such as adenovirus vectors, have high specific killing efficiency on gastric cancer and pancreatic cancer cells.
根据本发明的实施例，所述表达系统还可以进一步包括如下附加技术特征至少之一：According to the embodiment of the present invention, the expression system may further include at least one of the following additional technical features:
根据本发明的实施例，所述第一识别序列与所述第二识别序列分别独立地选自UAS,tetO以及dCas9的靶标序列至少之一。其中，所述UAS或tetO片段的重复次数可根据需要进行调整，如可选择2×UAS、3×UAS、4×UAS或5×UAS，5×tetO、6×tetO或7×tetO。According to an embodiment of the present invention, the first recognition sequence and the second recognition sequence are independently selected from at least one of UAS, tetO and dCas9 target sequences. Wherein, 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.
根据本发明的实施例，所述第一识别序列与所述第二识别序列为5×UAS。发明人在实验中意外地发现，使用癌特异启动子CEA368与癌特异microRNA mir-21及mir-143-3p调控的开关线路控制的溶瘤腺病毒、使用癌特异启动子CEA205与癌特异microRNA mir-21及mir-135a-5p调控的开关线路控制的溶瘤腺病毒、使用癌特异启动子CEA368与癌特异microRNA mir-21及mir-135a-5p调控的开关线路控制的溶瘤腺病毒、使用癌特异启动子CXCR4与癌特异microRNA mir-21及mir-135a-5p调控的开关线路控制的溶瘤腺病毒、使用癌特异启动子Survivin1与癌特异microRNA mir-21及mir-135a-5p调控的开关线路控制的溶瘤腺病毒可以特异性地杀伤胃癌细胞系AGS，5×UAS开关线路相较于4×UAS开关线路对胃正常细胞系GES1影响更小。According to an embodiment of the present invention, 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×UAS switch circuit, the 5×UAS switch circuit has less effect on the normal gastric cell line GES1.
根据本发明的实施例，所述转录激活因子为选自Gal4VP16、Gal4VP64、Gal4esn、dCas9-VP16、dCas9-VP64、dCas9-VPR、dCas9-VTR以及rtTA至少之一。According to an embodiment of the present invention, the transcription activator is at least one selected from Gal4VP16, Gal4VP64, Gal4esn, dCas9-VP16, dCas9-VP64, dCas9-VPR, dCas9-VTR and rtTA.
根据本发明的实施例，所述第一识别序列与所述第二识别序列分别独立地选自5×UAS, 7×tetO和dCas9的靶标序列至少之一。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述第一启动子与所述第二启动子分别独立地选自miniCMV、TATA box。According to an embodiment of the present invention, the first promoter and the second promoter are independently selected from miniCMV and TATA box.
根据本发明的实施例，所述第一调控蛋白和第二调控蛋白分别独立地选自Lacl、tetR、zinc finger(锌指)、TALE、KRAB、tetR-KRAB、、TALE-KRAB、dCas9-KRAB、miniCas9-KRAB、分割dCas9-KRAB至少之一。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述第一调控元件和所述第二调控元件分别独立地选自tetO、LacO、锌指蛋白目的序列(zinc finger target site)、TALE蛋白靶标序列、dCas9的靶标序列以及miniCas9的靶标序列至少之一。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述第一调控蛋白是LacI，所述第二调控元件包括多个重复的LacO序列，所述多个重复的LacO序列的至少之一设置在所述第二启动子的下游。LacI表达后，可特异性结合LacO序列，进而抑制第二启动子的功能。根据本发明的实施案例的LacI/LacO抑制系统，通过实验表明该系统可以有效地抑制启动子下游基因的表达。According to an embodiment of the present invention, 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. According to the 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.
根据本发明的实施例，所述第二调控蛋白是tetR-KRAB所述第一调控元件包括多个重复的tetO序列，所述多个重复的tetO序列的至少之一设置在所述第一启动子的下游。根据本发明的实施案例的tetR-KRAB/tetO抑制系统，可以有效的抑制启动子下游基因的表达。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述病毒复制包装蛋白、免疫效应因子可以以融合蛋白的形式存在。病毒复制包装蛋白可有效保证表达系统载体在宿主中的存活和复制；免疫效应因子的表达可有效激活机体的免疫系统，进而促进对胃癌细胞、胰腺癌细胞的免疫杀伤。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述病毒复制包装相关蛋白包括选自腺病毒E1基因,E1A基因，E1B基因，E2基因，E4基因至少之一。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述免疫效应因子包括选自拮抗PD-1基因的抑制序列、拮抗PD-L1基因的抑制序列、拮抗CTLA4基因的抑制序列、拮抗Tim-3基因的抑制序列、GM-CSF、IL-2、IL-12、IL-15至少之一。任选地，上述的免疫效应因子可以以融合蛋白的形式存在。According to an embodiment of the present invention, 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. Optionally, the aforementioned immune effector may exist in the form of a fusion protein.
根据本发明的实施例，所述目的蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目的蛋白与所述第一调控蛋白之间通过可切割的连接肽连接的。目的蛋白与第一调控蛋白在相同启动子下被调控表达，其表达后在连接肽处被切割，目的蛋白与第一调控蛋白分开，目的蛋白和第一调控蛋白相互独立地发挥功能。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述第一核酸分子与所述第二核酸分子负载在第一表达载体上，所述第三核酸分子、所述第四核酸分子、所述第五核酸分子以及所述第九核酸分子负载在第二表达载体上，所述第六核酸分子、所述第七核酸分子、所述第八核酸分子以及所述第 十核酸分子负载在第三表达载体上。第一、第二和第三表达载体作为表达系统的负载载体，实现目的蛋白在胃癌细胞、胰腺癌细胞的特异性表达。According to an embodiment of the present invention, the first nucleic acid molecule and the second nucleic acid molecule are loaded on a first expression vector, and 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, and 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.
所述表达载体的选择不受特别限制，只要能够实现表达系统在胃癌细胞、胰腺癌细胞的特异性发挥功能即可。根据本发明的具体实施例，所述第一表达载体、第二表达载体和第三表达载体分别独立地选自下列的至少之一：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. According to a specific embodiment of the present invention, the first expression vector, the second expression vector and the third expression vector are independently selected from at least one of the following:
质粒、病毒、稳定细胞系以及其他材料载体如纳米材料，脂质体，分子耦联载体、裸露DNA、染色体载体、多聚物。Plasmids, viruses, stable cell lines and other material carriers such as nanomaterials, liposomes, molecularly coupled carriers, naked DNA, chromosomal carriers, polymers.
根据本发明的实施例，所述病毒包括选自腺病毒、牛痘病毒、疱疹病毒、逆转录病毒的至少之一。According to an embodiment of the present invention, the virus includes at least one selected from the group consisting of adenovirus, vaccinia virus, herpes virus, and retrovirus.
根据本发明的实施例，所述第一表达载体、第二表达载体和第三表达载体构建负载在同一个载体上。需要说明的是，第一表达载体、第二表达载体和第三表达载体的连接顺序不受特别限制，只要不影响系统的生物学功能的实现即可。根据本发明的具体实施例，负载在同一表达载体上可以有效的解决多个大片段载体共转染效率极低的问题。According to an embodiment of the present invention, the first expression vector, the second expression vector and the third expression vector are constructed and loaded on the same vector. It should be noted that 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.
根据本发明的实施例，所述同一个载体为腺病毒载体。腺病毒作为基因治疗载体具有主范围广，对人致病性低、在增殖和非增殖细胞中感染和表达基因、滴度高、与人类基因同源、无插入致突变性、能在悬浮培养液中扩增、能同时表达多个基因的优点。According to an embodiment of the present invention, the same vector is an adenovirus vector. 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.
根据本发明的实施例，所述细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4 ×UAS；或所述细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS。根据本发明实施例的上述元件组成的表达系统控制的溶瘤腺病毒可以特异性地杀伤胃癌细胞，同时具有5×UAS的开关线路相较于具有4×UAS开关线路胃正常细胞的杀伤影响更小。According to an embodiment of the present invention, 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 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 And the second recognition sequence is 4×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 The sequence and the second recognition sequence are 4×UAS; or the cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first The recognition sequence and the second recognition sequence are 4×UAS; or the cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first microRNA is mir-135a-5p. 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. At the same time, the switch circuit with 5×UAS has a greater killing effect than normal gastric cells with 4×UAS switch circuit. small.
根据本发明的实施例，所述细胞特异性启动子为hMuc1，所述第一microRNA为mir-199a-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS。根据本发明实施例的上述元件组成的表达系统控制的溶瘤腺病毒可以特异性地杀伤胰腺癌细胞。According to an embodiment of the present invention, the cell-specific promoter is hMuc1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, and 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.
根据本发明的实施例，所述腺病毒载体携带具有SEQ ID NO：1～26任一所示核苷酸序列的核酸。According to an embodiment of the present invention, the adenovirus vector carries a nucleic acid having a nucleotide sequence shown in any one of SEQ ID NO: 1 to 26.
具有SEQ ID NO:1所述核苷酸序列的核酸编码由细胞特异性启动子CEA205、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000001
Figure PCTCN2020083823-appb-000001
Figure PCTCN2020083823-appb-000002
Figure PCTCN2020083823-appb-000002
Figure PCTCN2020083823-appb-000003
Figure PCTCN2020083823-appb-000003
Figure PCTCN2020083823-appb-000004
Figure PCTCN2020083823-appb-000004
具有SEQ ID NO:2所述核苷酸序列的核酸编码由细胞特异性启动子CEA205、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000005
Figure PCTCN2020083823-appb-000005
Figure PCTCN2020083823-appb-000006
Figure PCTCN2020083823-appb-000006
Figure PCTCN2020083823-appb-000007
Figure PCTCN2020083823-appb-000007
Figure PCTCN2020083823-appb-000008
Figure PCTCN2020083823-appb-000008
Figure PCTCN2020083823-appb-000009
Figure PCTCN2020083823-appb-000009
Figure PCTCN2020083823-appb-000010
Figure PCTCN2020083823-appb-000010
Figure PCTCN2020083823-appb-000011
Figure PCTCN2020083823-appb-000011
Figure PCTCN2020083823-appb-000012
Figure PCTCN2020083823-appb-000012
具有SEQ ID NO:3所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为LacI与tetR-KRAB、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000013
Figure PCTCN2020083823-appb-000013
Figure PCTCN2020083823-appb-000014
Figure PCTCN2020083823-appb-000014
Figure PCTCN2020083823-appb-000015
Figure PCTCN2020083823-appb-000015
Figure PCTCN2020083823-appb-000016
Figure PCTCN2020083823-appb-000016
Figure PCTCN2020083823-appb-000017
Figure PCTCN2020083823-appb-000017
具有SEQ ID NO:4所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000018
Figure PCTCN2020083823-appb-000018
Figure PCTCN2020083823-appb-000019
Figure PCTCN2020083823-appb-000019
Figure PCTCN2020083823-appb-000020
Figure PCTCN2020083823-appb-000020
Figure PCTCN2020083823-appb-000021
Figure PCTCN2020083823-appb-000021
Figure PCTCN2020083823-appb-000022
Figure PCTCN2020083823-appb-000022
Figure PCTCN2020083823-appb-000023
Figure PCTCN2020083823-appb-000023
Figure PCTCN2020083823-appb-000024
Figure PCTCN2020083823-appb-000024
Figure PCTCN2020083823-appb-000025
Figure PCTCN2020083823-appb-000025
具有SEQ ID NO:5所述核苷酸序列的核酸编码由细胞特异性启动子CXCR4、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为5×UAS的元件组成的表达系统(SEQ ID NO:5)。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.
Figure PCTCN2020083823-appb-000026
Figure PCTCN2020083823-appb-000026
Figure PCTCN2020083823-appb-000027
Figure PCTCN2020083823-appb-000027
Figure PCTCN2020083823-appb-000028
Figure PCTCN2020083823-appb-000028
Figure PCTCN2020083823-appb-000029
Figure PCTCN2020083823-appb-000029
具有SEQ ID NO:6所述核苷酸序列的核酸编码由细胞特异性启动子CXCR4、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000030
Figure PCTCN2020083823-appb-000030
Figure PCTCN2020083823-appb-000031
Figure PCTCN2020083823-appb-000031
Figure PCTCN2020083823-appb-000032
Figure PCTCN2020083823-appb-000032
Figure PCTCN2020083823-appb-000033
Figure PCTCN2020083823-appb-000033
Figure PCTCN2020083823-appb-000034
Figure PCTCN2020083823-appb-000034
Figure PCTCN2020083823-appb-000035
Figure PCTCN2020083823-appb-000035
Figure PCTCN2020083823-appb-000036
Figure PCTCN2020083823-appb-000036
Figure PCTCN2020083823-appb-000037
Figure PCTCN2020083823-appb-000037
具有SEQ ID NO:7所述核苷酸序列的核酸编码由细胞特异性启动子Survivin1、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000038
Figure PCTCN2020083823-appb-000038
Figure PCTCN2020083823-appb-000039
Figure PCTCN2020083823-appb-000039
Figure PCTCN2020083823-appb-000040
Figure PCTCN2020083823-appb-000040
Figure PCTCN2020083823-appb-000041
Figure PCTCN2020083823-appb-000041
Figure PCTCN2020083823-appb-000042
Figure PCTCN2020083823-appb-000042
具有SEQ ID NO:8所述核苷酸序列的核酸编码由细胞特异性启动子Survivin1、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000043
Figure PCTCN2020083823-appb-000043
Figure PCTCN2020083823-appb-000044
Figure PCTCN2020083823-appb-000044
Figure PCTCN2020083823-appb-000045
Figure PCTCN2020083823-appb-000045
Figure PCTCN2020083823-appb-000046
Figure PCTCN2020083823-appb-000046
Figure PCTCN2020083823-appb-000047
Figure PCTCN2020083823-appb-000047
Figure PCTCN2020083823-appb-000048
Figure PCTCN2020083823-appb-000048
Figure PCTCN2020083823-appb-000049
Figure PCTCN2020083823-appb-000049
Figure PCTCN2020083823-appb-000050
Figure PCTCN2020083823-appb-000050
具有SEQ ID NO:9所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000051
Figure PCTCN2020083823-appb-000051
Figure PCTCN2020083823-appb-000052
Figure PCTCN2020083823-appb-000052
Figure PCTCN2020083823-appb-000053
Figure PCTCN2020083823-appb-000053
Figure PCTCN2020083823-appb-000054
Figure PCTCN2020083823-appb-000054
具有SEQ ID NO:10所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000055
Figure PCTCN2020083823-appb-000055
Figure PCTCN2020083823-appb-000056
Figure PCTCN2020083823-appb-000056
Figure PCTCN2020083823-appb-000057
Figure PCTCN2020083823-appb-000057
Figure PCTCN2020083823-appb-000058
Figure PCTCN2020083823-appb-000058
Figure PCTCN2020083823-appb-000059
Figure PCTCN2020083823-appb-000059
Figure PCTCN2020083823-appb-000060
Figure PCTCN2020083823-appb-000060
Figure PCTCN2020083823-appb-000061
Figure PCTCN2020083823-appb-000061
Figure PCTCN2020083823-appb-000062
Figure PCTCN2020083823-appb-000062
具有SEQ ID NO:11所述核苷酸序列的核酸编码由细胞特异性启动子CEA205、第一 microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000063
Figure PCTCN2020083823-appb-000063
Figure PCTCN2020083823-appb-000064
Figure PCTCN2020083823-appb-000064
Figure PCTCN2020083823-appb-000065
Figure PCTCN2020083823-appb-000065
Figure PCTCN2020083823-appb-000066
Figure PCTCN2020083823-appb-000066
具有SEQ ID NO:12所述核苷酸序列的核酸编码由细胞特异性启动子CEA205、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000067
Figure PCTCN2020083823-appb-000067
Figure PCTCN2020083823-appb-000068
Figure PCTCN2020083823-appb-000068
Figure PCTCN2020083823-appb-000069
Figure PCTCN2020083823-appb-000069
Figure PCTCN2020083823-appb-000070
Figure PCTCN2020083823-appb-000070
Figure PCTCN2020083823-appb-000071
Figure PCTCN2020083823-appb-000071
Figure PCTCN2020083823-appb-000072
Figure PCTCN2020083823-appb-000072
Figure PCTCN2020083823-appb-000073
Figure PCTCN2020083823-appb-000073
Figure PCTCN2020083823-appb-000074
Figure PCTCN2020083823-appb-000074
具有SEQ ID NO:13所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000075
Figure PCTCN2020083823-appb-000075
Figure PCTCN2020083823-appb-000076
Figure PCTCN2020083823-appb-000076
Figure PCTCN2020083823-appb-000077
Figure PCTCN2020083823-appb-000077
Figure PCTCN2020083823-appb-000078
Figure PCTCN2020083823-appb-000078
具有SEQ ID NO:14所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000079
Figure PCTCN2020083823-appb-000079
Figure PCTCN2020083823-appb-000080
Figure PCTCN2020083823-appb-000080
Figure PCTCN2020083823-appb-000081
Figure PCTCN2020083823-appb-000081
Figure PCTCN2020083823-appb-000082
Figure PCTCN2020083823-appb-000082
Figure PCTCN2020083823-appb-000083
Figure PCTCN2020083823-appb-000083
Figure PCTCN2020083823-appb-000084
Figure PCTCN2020083823-appb-000084
Figure PCTCN2020083823-appb-000085
Figure PCTCN2020083823-appb-000085
具有SEQ ID NO:15所述核苷酸序列的核酸编码由细胞特异性启动子CXCR4、第一 microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000086
Figure PCTCN2020083823-appb-000086
Figure PCTCN2020083823-appb-000087
Figure PCTCN2020083823-appb-000087
Figure PCTCN2020083823-appb-000088
Figure PCTCN2020083823-appb-000088
Figure PCTCN2020083823-appb-000089
Figure PCTCN2020083823-appb-000089
具有SEQ ID NO:16所述核苷酸序列的核酸编码由细胞特异性启动子CXCR4、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000090
Figure PCTCN2020083823-appb-000090
Figure PCTCN2020083823-appb-000091
Figure PCTCN2020083823-appb-000091
Figure PCTCN2020083823-appb-000092
Figure PCTCN2020083823-appb-000092
Figure PCTCN2020083823-appb-000093
Figure PCTCN2020083823-appb-000093
Figure PCTCN2020083823-appb-000094
Figure PCTCN2020083823-appb-000094
Figure PCTCN2020083823-appb-000095
Figure PCTCN2020083823-appb-000095
Figure PCTCN2020083823-appb-000096
Figure PCTCN2020083823-appb-000096
Figure PCTCN2020083823-appb-000097
Figure PCTCN2020083823-appb-000097
具有SEQ ID NO:17所述核苷酸序列的核酸编码由细胞特异性启动子Survivin1、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000098
Figure PCTCN2020083823-appb-000098
Figure PCTCN2020083823-appb-000099
Figure PCTCN2020083823-appb-000099
Figure PCTCN2020083823-appb-000100
Figure PCTCN2020083823-appb-000100
Figure PCTCN2020083823-appb-000101
Figure PCTCN2020083823-appb-000101
具有SEQ ID NO:18所述核苷酸序列的核酸编码由细胞特异性启动子Survivin1、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000102
Figure PCTCN2020083823-appb-000102
Figure PCTCN2020083823-appb-000103
Figure PCTCN2020083823-appb-000103
Figure PCTCN2020083823-appb-000104
Figure PCTCN2020083823-appb-000104
Figure PCTCN2020083823-appb-000105
Figure PCTCN2020083823-appb-000105
Figure PCTCN2020083823-appb-000106
Figure PCTCN2020083823-appb-000106
Figure PCTCN2020083823-appb-000107
Figure PCTCN2020083823-appb-000107
Figure PCTCN2020083823-appb-000108
Figure PCTCN2020083823-appb-000108
具有SEQ ID NO:19所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-143-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成 的表达系统。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.
Figure PCTCN2020083823-appb-000109
Figure PCTCN2020083823-appb-000109
Figure PCTCN2020083823-appb-000110
Figure PCTCN2020083823-appb-000110
Figure PCTCN2020083823-appb-000111
Figure PCTCN2020083823-appb-000111
Figure PCTCN2020083823-appb-000112
Figure PCTCN2020083823-appb-000112
具有SEQ ID NO:20所述核苷酸序列的核酸编码由细胞特异性启动子CEA368、第一microRNA为mir-135a-5p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000113
Figure PCTCN2020083823-appb-000113
Figure PCTCN2020083823-appb-000114
Figure PCTCN2020083823-appb-000114
Figure PCTCN2020083823-appb-000115
Figure PCTCN2020083823-appb-000115
Figure PCTCN2020083823-appb-000116
Figure PCTCN2020083823-appb-000116
Figure PCTCN2020083823-appb-000117
Figure PCTCN2020083823-appb-000117
Figure PCTCN2020083823-appb-000118
Figure PCTCN2020083823-appb-000118
Figure PCTCN2020083823-appb-000119
Figure PCTCN2020083823-appb-000119
Figure PCTCN2020083823-appb-000120
Figure PCTCN2020083823-appb-000120
具有SEQ ID NO:21所述核苷酸序列的核酸编码由细胞特异性启动子hMUC1、第一microRNA为mir-199a-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000121
Figure PCTCN2020083823-appb-000121
Figure PCTCN2020083823-appb-000122
Figure PCTCN2020083823-appb-000122
Figure PCTCN2020083823-appb-000123
Figure PCTCN2020083823-appb-000123
Figure PCTCN2020083823-appb-000124
Figure PCTCN2020083823-appb-000124
Figure PCTCN2020083823-appb-000125
Figure PCTCN2020083823-appb-000125
具有SEQ ID NO:22所述核苷酸序列的核酸编码由细胞特异性启动子hMUC1、第一microRNA为mir-199a-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为Lacl、tetR-KRAB、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000126
Figure PCTCN2020083823-appb-000126
Figure PCTCN2020083823-appb-000127
Figure PCTCN2020083823-appb-000127
Figure PCTCN2020083823-appb-000128
Figure PCTCN2020083823-appb-000128
Figure PCTCN2020083823-appb-000129
Figure PCTCN2020083823-appb-000129
Figure PCTCN2020083823-appb-000130
Figure PCTCN2020083823-appb-000130
具有SEQ ID NO:23所述核苷酸序列的核酸编码由细胞特异性启动子hMUC1、第一microRNA为mir-199a-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为4×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000131
Figure PCTCN2020083823-appb-000131
Figure PCTCN2020083823-appb-000132
Figure PCTCN2020083823-appb-000132
Figure PCTCN2020083823-appb-000133
Figure PCTCN2020083823-appb-000133
Figure PCTCN2020083823-appb-000134
Figure PCTCN2020083823-appb-000134
Figure PCTCN2020083823-appb-000135
Figure PCTCN2020083823-appb-000135
Figure PCTCN2020083823-appb-000136
Figure PCTCN2020083823-appb-000136
Figure PCTCN2020083823-appb-000137
Figure PCTCN2020083823-appb-000137
Figure PCTCN2020083823-appb-000138
Figure PCTCN2020083823-appb-000138
具有SEQ ID NO:24所述核苷酸序列的核酸编码由细胞特异性启动子hMUC1、第一microRNA为mir-199a-3p、第二microRNA为mir-21、转录激活因子为Gal4VP16、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000139
Figure PCTCN2020083823-appb-000139
Figure PCTCN2020083823-appb-000140
Figure PCTCN2020083823-appb-000140
Figure PCTCN2020083823-appb-000141
Figure PCTCN2020083823-appb-000141
Figure PCTCN2020083823-appb-000142
Figure PCTCN2020083823-appb-000142
Figure PCTCN2020083823-appb-000143
Figure PCTCN2020083823-appb-000143
Figure PCTCN2020083823-appb-000144
Figure PCTCN2020083823-appb-000144
Figure PCTCN2020083823-appb-000145
Figure PCTCN2020083823-appb-000145
Figure PCTCN2020083823-appb-000146
Figure PCTCN2020083823-appb-000146
具有SEQ ID NO:25所述核苷酸序列的核酸编码由细胞特异性启动子hMUC1、第一microRNA为mir-199a-3p、第二microRNA为mir-21、转录激活因子为分割dCas9、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000147
Figure PCTCN2020083823-appb-000147
Figure PCTCN2020083823-appb-000148
Figure PCTCN2020083823-appb-000148
Figure PCTCN2020083823-appb-000149
Figure PCTCN2020083823-appb-000149
Figure PCTCN2020083823-appb-000150
Figure PCTCN2020083823-appb-000150
Figure PCTCN2020083823-appb-000151
Figure PCTCN2020083823-appb-000151
Figure PCTCN2020083823-appb-000152
Figure PCTCN2020083823-appb-000152
Figure PCTCN2020083823-appb-000153
Figure PCTCN2020083823-appb-000153
Figure PCTCN2020083823-appb-000154
Figure PCTCN2020083823-appb-000154
Figure PCTCN2020083823-appb-000155
Figure PCTCN2020083823-appb-000155
具有SEQ ID NO:26所述核苷酸序列的核酸编码由细胞特异性启动子hMUC1、第一 microRNA为mir-199a-3p、第二microRNA为mir-21、转录激活因子为分割dCas9、第一调控蛋白和第二调控蛋白分别为TALE蛋白、第一和第二识别序列为5×UAS的元件组成的表达系统。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.
Figure PCTCN2020083823-appb-000156
Figure PCTCN2020083823-appb-000156
Figure PCTCN2020083823-appb-000157
Figure PCTCN2020083823-appb-000157
Figure PCTCN2020083823-appb-000158
Figure PCTCN2020083823-appb-000158
Figure PCTCN2020083823-appb-000159
Figure PCTCN2020083823-appb-000159
Figure PCTCN2020083823-appb-000160
Figure PCTCN2020083823-appb-000160
Figure PCTCN2020083823-appb-000161
Figure PCTCN2020083823-appb-000161
Figure PCTCN2020083823-appb-000162
Figure PCTCN2020083823-appb-000162
Figure PCTCN2020083823-appb-000163
Figure PCTCN2020083823-appb-000163
Figure PCTCN2020083823-appb-000164
Figure PCTCN2020083823-appb-000164
根据本发明的实施例，所述腺病毒是通过如下方式获得的：所述的腺病毒载体去除了与腺病毒复制包装相关的E1基因和部分E3基因，E1A基因通过逐级Golden Gate的方法构建到基因线路中，最终通过Gateway或Gibson的方式将基因线路插入到腺病毒载体中。上述获得腺病毒的方式实现了复杂、大片段溶瘤腺病毒载体的快速改造。具体构建方法可参考201780002478.X。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述腺病毒载体为去除E1基因与部分E3基因的的B、C亚型腺病毒。According to an embodiment of the present invention, the adenovirus vector is an adenovirus of subtypes B and C with E1 gene and part of E3 gene removed.
根据本发明的实施例，所述腺病毒载体为去除E1基因与部分E3基因的5、11、12、34或35型腺病毒。According to an embodiment of the present invention, 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.
根据本发明的实施例，SEQ ID NO:1～26任一所示的核苷酸序列在所述5型腺病毒载体的插入位点为E1基因区域、E3基因区域或E4基因区域。例如，去除E1基因与部分E3基因5型腺病毒载体的图谱可参见图13，其序列如SEQ ID NO:27所示，其插入位点可以选择在该序列的第458个碱基后第459个碱基前。According to an embodiment of the present invention, 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. For example, 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.
Figure PCTCN2020083823-appb-000165
Figure PCTCN2020083823-appb-000165
Figure PCTCN2020083823-appb-000166
Figure PCTCN2020083823-appb-000166
Figure PCTCN2020083823-appb-000167
Figure PCTCN2020083823-appb-000167
Figure PCTCN2020083823-appb-000168
Figure PCTCN2020083823-appb-000168
Figure PCTCN2020083823-appb-000169
Figure PCTCN2020083823-appb-000169
Figure PCTCN2020083823-appb-000170
Figure PCTCN2020083823-appb-000170
Figure PCTCN2020083823-appb-000171
Figure PCTCN2020083823-appb-000171
Figure PCTCN2020083823-appb-000172
Figure PCTCN2020083823-appb-000172
Figure PCTCN2020083823-appb-000173
Figure PCTCN2020083823-appb-000173
Figure PCTCN2020083823-appb-000174
Figure PCTCN2020083823-appb-000174
Figure PCTCN2020083823-appb-000175
Figure PCTCN2020083823-appb-000175
Figure PCTCN2020083823-appb-000176
Figure PCTCN2020083823-appb-000176
Figure PCTCN2020083823-appb-000177
Figure PCTCN2020083823-appb-000177
Figure PCTCN2020083823-appb-000178
Figure PCTCN2020083823-appb-000178
Figure PCTCN2020083823-appb-000179
Figure PCTCN2020083823-appb-000179
Figure PCTCN2020083823-appb-000180
Figure PCTCN2020083823-appb-000180
Figure PCTCN2020083823-appb-000181
Figure PCTCN2020083823-appb-000181
Figure PCTCN2020083823-appb-000182
Figure PCTCN2020083823-appb-000182
在本发明的第二方面，本发明提出了一种重组病毒。根据本发明的实施例，所述重组病毒包括：第一核酸分子，所述第一核酸分子含有肿瘤细胞特异性启动子；第二核酸分子，所述第二核酸分子与所述第一核酸分子可操作地连接，所述第二核酸分子编码转录激活因子，所述转录激活因子为Gal4VP16；第三核酸分子，所述第三核酸分子含有所述转录激活因子的第一识别序列；第四核酸分子，所述第四核酸分子与所述第三核酸分子可操作地连接，所述第四核酸分子含有第一启动子和第一调控元件，所述第一启动子为miniCMV，所述第一调控元件包括多个重复的tetO序列，所述多个重复的tetO序列的至少之一设置在所述第一启动子的下游；第五核酸分子，所述第五核酸分子与第四核酸分子可操作地连接，所述第五核酸分子编码第一调控蛋白，所述第一调控蛋白为LacI；所述第五核酸分子进一步包括编码所述目的蛋白的序列，并且所述目的蛋白包括病毒复制蛋白、免疫效应因子，所述免疫效应因子是以单独或融合蛋白的形式表达的，并且所述毒复制蛋白和所述效应因子之间通过可切割的连接肽连接的，所述目的蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目的蛋白与所述第一调控蛋白之间通过可切割的连接肽连接的；第六核酸分子，所述第六核酸分子含有所述转录激活因子的第二识别序列；第七核酸分子，所述第七核酸分子与所述第六核酸分子可操作地连接，所述第七核酸分子含有第二启动子和第二调控元件，所述第二启动子为miniCMV，所述第二调控元件包括多个重复的LacO序列，所述多个重复的LacO序列的至少之一设置在所述第二启动子的下游；第八核酸分子，所述第八核酸分子与第七核酸分子可操作地连接，并且所述第八核酸分子编码第二调控蛋白,所述第二调控蛋白为tetR-KRAB；第九核酸分子，所述第九核酸分子与所述第五核酸分子可操作地连接，所述第九核酸分子被配置为条件性抑制所述第一调控蛋白的表达，所述第九核酸分子含有被第一microRNA特异性识别的核酸序列，所述第一microRNA为正常细胞特异性 microRNA；以及第十核酸分子，所述第十核酸分子与所述第八核酸分子可操作地连接，所述第十核酸分子被配置为条件性抑制所述第二调控蛋白的表达，所述第十核酸分子含有被第二microRNA特异性识别的核酸序列，所述第二microRNA为肿瘤细胞特异性microRNA，其中，所述第一调控元件适于通过结合所述第二调控蛋白抑制所述第一启动子的功能，所述第二调控元件适于通过结合所述第一调控蛋白抑制所述第二启动子的功能；所述肿瘤细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述肿瘤细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述肿瘤细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或所述肿瘤细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述肿瘤细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述肿瘤细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或所述肿瘤细胞特异性启动子为hMuc1，所述第一microRNA为mir-199a-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS。利用根据本申请实施例的重组病毒，在肿瘤细胞特异性启动子和第九核酸分子、第十核酸分子的共同调控下，第一调控蛋白LacI、目的蛋白在胃癌、胰腺癌细胞中特异性表达，第二调控蛋白tetR-KRAB在胃癌、胰腺癌细胞中特异性不表达或低表达，进而tetR-KRAB介导的第一启动子miniCMV的抑制机制解除，第一调控蛋白LacI、目的蛋白在第一启动子miniCMV的启动调控下有效表达，LacI介导的抑制机制有效抑制了第二启动子miniCMV的功能，tetR-KRAB的表达进一步受到抑制。进而，利用根据本发明实施例的重组病毒，可实现蛋白在胃癌、胰腺癌细胞中更加特异性的表达(如目的蛋白或LacI)或不表达(如tetR-KRAB)，在胃癌、胰腺癌细胞中的目的蛋白表达 表达效率和特异性高。根据本发明实施例的重组病毒可实现对胃癌、胰腺癌细胞的特异、高效、安全的杀伤。In the second aspect of the present invention, the present invention proposes a recombinant virus. According to an embodiment of the present invention, 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 arranged downstream of the first promoter; a fifth nucleic acid molecule, the fifth nucleic acid molecule and the fourth nucleic acid molecule can be Operatively connected, the fifth nucleic acid molecule encodes a first regulatory protein, the first regulatory protein is LacI; the fifth nucleic acid molecule further includes a sequence encoding the target protein, and the target protein includes a viral replication protein , Immune effector, said immune effector is expressed in the form of a separate or fusion protein, and the virus replication protein and said effector are connected by a cleavable linking peptide, said target protein and said The first regulatory protein is 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 sixth nucleic acid molecule, the sixth nucleic acid molecule contains the The second recognition sequence of a transcription activator; a seventh nucleic acid molecule, said seventh nucleic acid molecule is operably linked to said sixth nucleic acid molecule, said seventh nucleic acid molecule contains a second promoter and a second regulatory element, so The second promoter is miniCMV, the second regulatory element includes a plurality of repeated LacO sequences, at least one of the plurality of repeated LacO sequences is arranged downstream of the second promoter; an eighth nucleic acid molecule, The eighth nucleic acid molecule is operably linked to the seventh nucleic acid molecule, and the eighth nucleic acid molecule encodes a second regulatory protein, and the second regulatory protein is tetR-KRAB; a ninth nucleic acid molecule, the ninth nucleic acid The molecule is operably connected to the fifth nucleic acid molecule, the ninth nucleic acid molecule is configured to conditionally inhibit the expression of the first regulatory protein, and the ninth nucleic acid molecule contains a nucleic acid specifically recognized by the first microRNA Sequence, the first microRNA is a normal cell-specific microRNA; and a tenth nucleic acid molecule, the tenth nucleic acid molecule is operably linked to the eighth nucleic acid molecule, and the tenth nucleic acid molecule is configured to conditionally inhibit For the expression of the second regulatory protein, the tenth nucleic acid molecule contains a nucleic acid sequence specifically recognized by a second microRNA, the second microRNA is a tumor cell-specific microRNA, and the first regulatory element is suitable for passing through Combining with the second regulatory protein to inhibit the function of the first promoter The second regulatory element is suitable for inhibiting the function of the second promoter by binding to the first regulatory protein; the tumor cell-specific promoter is CEA205, and the first microRNA is mir-135a-5p, The second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 5×UAS; or the tumor cell-specific promoter is CEA368, and the first microRNA is mir-135a- 5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 5×UAS; or the tumor cell-specific promoter is CXCR4, and the first microRNA is mir- 135a-5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 5×UAS; or the tumor cell-specific promoter is Survivin1, and the first microRNA is mir-135a-5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 5×UAS; or the tumor cell-specific promoter is CEA368, the first The microRNA is mir-143-3p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 5×UAS; or the tumor cell-specific promoter is CEA205, the The first microRNA is mir-135a-5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 4×UAS; or the tumor cell-specific promoter is CEA368, The first microRNA is mir-135a-5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 4×UAS; or the tumor cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 4×UAS; or the tumor cell-specific activation The sub is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, the first recognition sequence and the second recognition sequence are 4×UAS; or the tumor cell-specific The sex promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS; or the tumor The cell-specific promoter is hMuc1, the first microRNA is mir-199a-3p, and the second microRNA is mir-2 1. The first identification sequence and the second identification sequence are 4×UAS. Using the recombinant virus according to the embodiments of the application, 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. Furthermore, by using the recombinant virus according to the embodiments of the present invention, more specific expression of the protein (such as the target protein or LacI) or no expression (such as tetR-KRAB) in gastric cancer and pancreatic cancer cells can be achieved. 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.
根据本发明的实施例，上述重组病毒还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the aforementioned recombinant virus may further include at least one of the following additional technical features:
根据本发明的实施例，所述重组病毒为选自逆转录病毒、腺病毒、疱疹病毒、牛痘病毒的至少之一。According to an embodiment of the present invention, the recombinant virus is at least one selected from retrovirus, adenovirus, herpes virus, and vaccinia virus.
根据本发明的实施例，所述重组病毒为腺病毒。如前所述，腺病毒作为基因治疗载体具有宿主范围广，对人致病性低、在增殖和非增殖细胞中感染和表达基因、滴度高、与人类基因同源、无插入致突变性、能在悬浮培养液中扩增、能同时表达多个基因的优点。According to an embodiment of the present invention, the recombinant virus is an adenovirus. As mentioned above, 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.
根据本发明的实施例，所述免疫效应因子包括选自拮抗PD-1基因的抑制序列、拮抗PD-L1基因的抑制序列、拮抗CTLA4基因的抑制序列、拮抗Tim-3基因的抑制序列、IL-2、IL-15、IL-12、GM-CSF至少之一。任选地，所述免疫效应因子可以以融合蛋白的形式存在。According to an embodiment of the present invention, 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. Optionally, the immune effector may be in the form of a fusion protein.
在本发明的第三方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞含有前面所述的表达系统。根据本发明实施例的重组细胞可有效激活人体的系统性的免疫反应，特异性攻击胃癌、胰腺癌细胞，安全性高，特异性强。In the third aspect of the present invention, the present invention proposes a recombinant cell. According to an embodiment of the present invention, 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.
根据本发明的实施例，上述重组细胞还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the aforementioned recombinant cell may further include at least one of the following additional technical features:
根据本发明的实施例，所述表达系统的至少一部分整合于所述重组细胞的基因组中。表达系统随着重组细胞基因组的复制而复制，表达系统对目的蛋白的表达调控持续而有效。According to an embodiment of the present invention, 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.
在本发明的第四方面，本发明提出了前面所述的表达系统、前面所述的重组病毒、前面所述的重组细胞在制备药物中的用途，所述药物用于治疗胃癌或胰腺癌。In the fourth aspect of the present invention, 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.
在本发明的第五方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包含前面所述的重组病毒或前面所述的重组细胞。根据本发明实施例的药物组合物对胃癌或胰腺癌的治疗效果显著。In the fifth aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, 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.
根据本发明的实施例，上述药物组合物还可以进一步包括药学上可接受的辅料。According to an embodiment of the present invention, the aforementioned pharmaceutical composition may further include pharmaceutically acceptable excipients.
根据本发明的实施例，所述药物组合物进一步包括其他治疗胃癌的药物。According to an embodiment of the present invention, the pharmaceutical composition further includes other drugs for treating gastric cancer.
根据本发明的实施例，所述其他治疗胃癌的药物包括选自Pembrolizumab、Ogivri、Everolimus、Lanreotide、Ramucirumab、Apatinib、Trastuzumab的至少之一。According to an embodiment of the present invention, the other drugs for treating gastric cancer include at least one selected from Pembrolizumab, Ogivri, Everolimus, Lanreotide, Ramucirumab, Apatinib, and Trastuzumab.
根据本发明的实施例，所述药物组合物进一步包括其他治疗胰腺癌的药物。According to an embodiment of the present invention, the pharmaceutical composition further includes other drugs for treating pancreatic cancer.
根据本发明的实施例，所述其他治疗胰腺癌的药物包括选自Lanreotide、Abraxane、Olaparib、Afinitor、Erlotinib、Everolimus、5-FU、Gemzar、Sunitinib、Onivyde、Gemzar的至少之一。According to an embodiment of the present invention, 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.
本发明的药物组合物可以在治疗或预防胃癌、胰腺癌时被给药。The pharmaceutical composition of the present invention can be administered when treating or preventing gastric cancer and pancreatic cancer.
在本文中所使用的术语“给药”指将预定量的物质通过某种适合的方式引入病人。本发明的药物组合物可以通过任何常见的途径被给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。优选地，本发明的组合物可以注射制剂被给药。此外，本发明的药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。The term "administration" as used herein 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. However, due to oral administration, the active ingredient of the oral administration composition should be coated or formulated to prevent its degradation in the stomach. Preferably, the composition of the present invention may be administered as an injection formulation. In addition, the pharmaceutical composition of the present invention can be administered using a specific device that delivers the active ingredient to the target cell.
本发明的药物组合物的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。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. According to some embodiments of the present invention, 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 .
术语“治疗有效量”是指化合物足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。例如，在胃癌或胰腺癌的治疗中，减少、预防、延缓、抑制或阻滞疾病或病症的任何症状的药物或化合物应当是治疗有效的。治疗有效量的药物或化合物不需要治愈疾病或病症，但将为疾病或病症提供治疗，使得个体的疾病或病症的发作被延缓、阻止或预防，或者疾病或病症的症状得以缓解，或者疾病或病症的期限被改变，或者例如疾病或病症变得不严重，或者加速康复。The term "therapeutically 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. For example, in the treatment of gastric cancer or pancreatic cancer, 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.
术语“治疗”用于指获得期望的药理学和/或生理学效果。所述效果就完全或部分预防疾病或其症状而言可以是预防性的，和/或就部分或完全治愈疾病和/或疾病导致的不良作用而言可以是治疗性的。本文使用的“治疗”涵盖哺乳动物、特别是人的疾病(主要指胃癌或胰腺癌)的治疗，包括：(a)在容易患病但是尚未确诊得病的个体中预防疾病(例如预防胃癌或胰腺癌)或病症发生；(b)抑制疾病，例如阻滞疾病发展；或(c)缓解疾病，例如减轻与疾病相关的症状。本文使用的“治疗”涵盖将药物或化合物给予个体以治疗、治愈、缓解、改善、减轻或抑制个体的疾病的任何用药，包括但不限于将含本文所述的药物给予有需要的个体。The term "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" as used herein 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" as used herein 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.
根据本发明的实施例，本发明的药物组合物可与常规治疗方法和/或疗法相结合使用，或者可与常规治疗方法和/或疗法分开使用。当本发明的药物在采用与其它药物的联合疗法中给药时，它们可序贯地或同时地给予个体。或者，本发明的药物组合物可包含本发明的重组病毒或药学上可接受的赋形剂以及本领域已知的其它治疗药或预防药的组合。According to the embodiments of the present invention, 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. When the drugs of the present invention are administered in combination therapy with other drugs, they can be administered to the individual sequentially or simultaneously. Alternatively, 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.
需要说明的是，如无特别说明，本申请所述的融合蛋白是指在同一启动子调控下共转录的蛋白，包括蛋白间无连接的融合蛋白，或以其他连接肽(如GGGS或2A序列)连接的融合蛋白。It should be noted that, unless otherwise specified, 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.
如无特别说明，本申请所述的闭合回路或表达系统的“翻转能力”是指受第一microRNA和第二microRNA输入信号分别控制时输出水平的差异，具体体现在目的蛋白(包括选自病毒复制包装蛋白、免疫效应因子的至少之一)表达水平的差异。Unless otherwise specified, 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.
本申请所述的“几×序列”是指几段重复的序列顺序连接，重复序列之间没有间隔碱基，例如，“5×UAS”是指5段重复的UAS顺利连接所组成的序列，“6×tetO”是指6段重复的tetO顺序连接所组成的序列。The "several × sequence" mentioned in this application refers to the sequential connection of several repeated sequences with no intervening bases between the repeated sequences. For example, "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.
附图说明Description of the drawings
图1为根据本发明实施例的靶向特定肿瘤的溶瘤腺病毒构建与测试流程示意图；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;
图2为根据本发明实施例的不同癌特异启动子在胃癌与胃正常细胞系中的表达测试；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;
图3为根据本发明实施例的不同癌特异启动子在胃癌与胃正常细胞系中翻转开关线路的测试；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;
图4为根据本发明实施例的不同microRNA靶位点在胃癌与胃正常细胞系中的抑制效率测试；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;
图5A～5J为根据本发明实施例的不同腺病毒在胃癌与正常细胞系中的杀伤能力测试；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;
图6为根据本发明实施例的不同癌特异启动子在乳腺癌与乳腺正常细胞系中的表达测试；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;
图7为根据本发明实施例的不同癌特异启动子在乳腺癌与乳腺正常细胞系中翻转开关线路的测试；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;
图8为根据本发明实施例的不同microRNA靶位点在乳腺癌与乳腺正常细胞系中的抑制效率测试。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.
图9A～9D为根据本发明实施例的不同腺病毒在乳腺癌与正常细胞系中的杀伤能力测试；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;
图10为不同癌特异启动子在胰腺癌与胰腺正常细胞系中的表达测试；Figure 10 shows the expression test of different cancer-specific promoters in pancreatic cancer and normal pancreatic cell lines;
图11为根据本发明实施例的不同癌特异启动子在胰腺癌与胰腺正常细胞系中翻转开关线路的测试；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;
图12为根据本发明实施例的不同腺病毒在胰腺癌与正常细胞系中的杀伤能力测试；以及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; and
图13为根据本发明实施例的5型腺病毒载体的图谱。Figure 13 is a map of a type 5 adenovirus vector according to an embodiment of the present invention.
具体实施方式Detailed ways
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描 述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention. Where specific techniques or conditions are not indicated in the examples, the procedures shall be carried out in accordance with the techniques or conditions described in the literature in the field or in accordance with the product specification. The reagents or instruments used without the manufacturer's indication are all conventional products that are commercially available.
在以下实施例中，涉及的构建靶向特定肿瘤的溶瘤腺病毒的方法以及测试流程可参照示意图，图1；所涉及的病毒代号如表1所示。In the following examples, 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.
表1：Table 1:
Figure PCTCN2020083823-appb-000183
Figure PCTCN2020083823-appb-000183
实施例1靶向胃癌的溶瘤腺病毒的构建与功能验证Example 1 Construction and functional verification of oncolytic adenovirus targeting gastric cancer
实验一experiment one
测定癌特异性启动子CEA205、CEA368、cerbB2、COX2、CXCR4、hMUC1、Survivin1与Survivin3在胃癌细胞系AGS与胃正常细胞系GES1中的表达强度与特异性。The expression intensity and specificity of cancer-specific promoters CEA205, CEA368, cerbB2, COX2, CXCR4, hMUC1, Survivin1 and Survivin3 in gastric cancer cell line AGS and gastric normal cell line GES1 were determined.
将CEA205-Gal4VP16的质粒、UAS-EYFP质粒、hEF1a-EBFP2质粒共转染胃癌细胞系AGS与胃正常细胞系GES1(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分 析，检测EYFP与EBFP2荧光强度。归一化报告基因表达水平＝(实验组EYFP荧光强度/实验组EBFP2荧光强度)/(对照组EYFP荧光强度/对照组EBFP2荧光强度)。The 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).
用CEA368-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用cerbB2-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用COX2-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用CXCR4-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用hMUC1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用Survivin1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用Survivin3-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。Use the 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. Use Survivin1-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid to perform the above steps. Use Survivin3-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.
归一化报告基因表达水平见图2。各癌特异启动子在胃正常细胞系GES1中表达量均很低，而在胃癌细胞系AGS中，CEA205、CEA368、CXCR4与Survivin1表达量较高。结果表明：癌特异启动子可以特异性地在胃癌细胞系中启动Gal4VP16的表达并激活UAS下游的报告基因。CEA205、CEA368、CXCR4与Survivin1有较好的表达强度与特异性。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.
实验二Experiment two
在实验一的基础上，测定具有较好表达强度与特异性的CEA205、CEA368、CXCR4与Survivin1启动5×UAS或4×UAS开关线路的翻转能力。On the basis of experiment 1, the ability of CEA205, CEA368, CXCR4 and Survivin1 with better expression intensity and specificity to initiate 5×UAS or 4×UAS switch circuits was determined.
将CEA205-Gal4VP16质粒、5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒、5×UAS-LacO-TetRKrab-FF5质粒、CAG-EYFP质粒、pDT7004质粒共转染胃癌细胞系AGS与胃正常细胞系GES1(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分析，检测EYFP与EBFP2荧光强度，表示无输入信号时开关线路的表达水平。报告基因表达水平＝EBFP2荧光强度/EYFP荧光强度。The CEA205-Gal4VP16 plasmid, 5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 5×UAS-LacO-TetRKrab-FF5 plasmid, CAG-EYFP plasmid, and pDT7004 plasmid were co-transfected into the gastric cancer cell line AGS With the normal gastric cell line GES1 (100ng of each plasmid transfected in each well), flow cytometry analysis was performed 48 hours after transfection to detect the fluorescence intensity of EYFP and EBFP2, which indicates the expression level of the switch circuit when there is no input signal. Reporter gene expression level = EBFP2 fluorescence intensity/EYFP fluorescence intensity.
用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤，表示一侧输入信号时开关线路的表达水平；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤，表示另一侧输入信号时开关线路的表达水平。Use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps, to indicate the expression level of the switch circuit when one side of the signal is input; use U6-shRNA-FF5-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps, Indicates the expression level of the switch circuit when the signal is input from the other side.
用4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4×UAS-LacO-TetRKrab-FF5 plasmid instead After 5×UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps; use U6-shRNA-FF5-CMV-iRFP plasmid instead of pDT7004 plasmid, Perform the above steps.
用CEA368-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CEA368-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CEA368-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 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.
用CXCR4-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CXCR4-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CXCR4-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use CXCR4-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.
用Survivin1-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with Survivin1-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用Survivin1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use Survivin1-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.
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CAG-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进 行上述步骤。Use 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.
报告基因表达水平见图3。与实验一结果一致，被测试的所有启动子在胃正常细胞系GES1中表达量均很低，而在胃癌细胞系AGS中均可以启动开关线路翻转。在胃癌细胞系AGS中CEA368与Survivin1表达量略高于CEA205与CXCR4，而5×UAS开关线路表达强度略高于4×UAS开关线路。结果表明：被测试的所有启动子均可以特异性地在胃癌细胞系AGS中启动4×UAS或5×UAS开关线路翻转。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.
实验三Experiment Three
测定癌特异性microRNA mir-143-3p、mir-145-5p、mir-551b-3p、mir-1-3p、mir-133a-3p、mir-133b-3p、mir-139-3p、mir-139-5p、mir-145-3p、mir-204-5p、mir-195-3p、mir-935、mir-135a-5p、mir-135b-5p、mir-135b-3p、mir-196a-3p与mir-21在胃癌细胞系AGS与胃正常细胞系GES1中对各靶位点的抑制强度。Measure cancer-specific microRNA mir-143-3p, mir-145-5p, mir-551b-3p, mir-1-3p, mir-133a-3p, mir-133b-3p, mir-139-3p, mir-139 -5p, mir-145-3p, mir-204-5p, mir-195-3p, mir-935, mir-135a-5p, mir-135b-5p, mir-135b-3p, mir-196a-3p and mir -21 The inhibitory strength of each target site in the gastric cancer cell line AGS and the gastric normal cell line GES1.
将CMV-EYFP-T143 3p x4质粒、CMV-EBFP2质粒、pDT7004质粒共转染胃癌细胞系AGS与胃正常细胞系GES1(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分析，检测EYFP与EBFP2荧光强度。设置用CMV-EYFP质粒代替CMV-EYFP-T143 3p x4质粒的对照处理。归一化报告基因表达水平＝(实验组EBFP2荧光强度/实验组EYFP荧光强度)/(对照组EBFP2荧光强度/对照组EYFP荧光强度)。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. Set up a control treatment with CMV-EYFP plasmid instead of CMV-EYFP-T143 3p 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).
用CMV-EYFP-T145 5p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T551 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T1 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T133a 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T133b 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T139 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T139 5p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T145 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T204 5p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T195 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T935x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T135a 5p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T135b 5p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T135b 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T196a 5p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用CMV-EYFP-T196a 3p x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。用 CMV-EYFP-T21x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T145 5p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T551 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T1 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T133a 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T133b 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T139 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T139 5p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T145 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T204 5p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T195 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T935x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T135a 5p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T135b 5p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T135b 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T196a 5p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T196a 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T21x4 plasmid, perform the above steps.
归一化报告基因表达水平见图4。mir-21可以通过靶位点特异性地在胃癌细胞系AGS中抑制报告基因表达，并有很高的抑制强度。mir-143-3p与mir-135a-5p可以通过靶位点特异性地在胃正常细胞系GES1中抑制报告基因表达，并有一定地抑制强度。结果表明：癌特异高表达或低表达的microRNA可以通过靶位点特异性地在胃癌细胞系AGS或胃正常细胞系GES1中抑制报告基因表达。癌特异高表达microRNA mir-21、癌特异低表达microRNA mir-143-3p与mir-135a-5p有较好的抑制强度。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.
实验四Experiment Four
实验一、二、三的基础上，构建使用CEA205/CEA368/CXCR4/Survivin1启动子、mir-21与mir-135a-5p/mir-143-3p调控的4×UAS或5×UAS开关线路控制的溶瘤腺病毒，测试对胃癌细胞系AGS、胃正常细胞系GES1、肝正常细胞系Chang与L02的杀伤能力。On the basis of experiments one, two and three, construct the 4×UAS or 5×UAS switch circuit controlled by CEA205/CEA368/CXCR4/Survivin1 promoter, mir-21 and mir-135a-5p/mir-143-3p Oncolytic adenovirus, test the killing ability against gastric cancer cell line AGS, normal gastric cell line GES1, normal liver cell line Chang and L02.
在96孔板中每孔接种数量为1e4的细胞。接种24小时后分别加入感染复数为1、10、20、50、100、200、500的A1病毒，设置不加入病毒的空白对照。病毒侵染6天后使用MTS法检测细胞存活率。细胞存活率＝实验组MTS检测值/对照组MTS检测值。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.
使用A2病毒代替A1病毒，进行上述步骤。使用A3病毒代替A1病毒，进行上述步骤。使用A4病毒代替A1病毒，进行上述步骤。使用A5病毒代替A1病毒，进行上述步骤。使用A6病毒代替A1病毒，进行上述步骤。使用A7病毒代替A1病毒，进行上述步骤。使用A8病毒代替A1病毒，进行上述步骤。使用A9病毒代替A1病毒，进行上述步骤。使用A10病毒代替A1病毒，进行上述步骤。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. Use A9 virus instead of A1 virus to perform the above steps. Use A10 virus instead of A1 virus to perform the above steps.
细胞存活率见图5A～5J。与实验一、二、三结果一致，使用癌特异启动子CEA205/CEA368/CXCR4/Survivin1与癌特异microRNA mir-21及mir-135a-5p/mir-143-3p调控的开关线路控制的溶瘤腺病毒可以特异性地杀伤胃癌细胞系AGS，而对胃正常细胞系GES1及肝正常细胞系Chang、L02杀伤作用不明显。5×UAS开关线路控制的溶瘤腺病毒A1在感染复数约10～20间时就表现出明显的杀伤特异性，而A2、A4、A5与A1杀伤效果相近。4×UAS开关线路控制的溶瘤腺病毒A10与A1杀伤效果相近。结果表明：使用癌特异启动子CEA205/CEA368/Survivin1与癌特异microRNA mir-21及mir-135a-5p/mir-143-3p调控的开关线路控制的溶瘤腺病毒可以特异性地杀伤胃癌细胞系AGS，5×UAS开关线路相较于4×UAS开关线路效果更好。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.
实施例2靶向乳腺癌的溶瘤腺病毒的构建与功能验证Example 2 Construction and functional verification of oncolytic adenovirus targeting breast cancer
实验一experiment one
结合实施例1实验结果，测定癌特异性启动子CEA205、CEA368、CXCR4、hMUC1 与Survivin1在乳腺癌细胞系MCF7、MDA-MB-231与乳腺正常细胞系MCF10A中的表达强度与特异性。Combined with the experimental results of Example 1, 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.
将CEA205-Gal4VP16的质粒、UAS-EYFP质粒、CMV-EBFP2质粒共转染乳腺癌细胞系MCF7、MDA-MB-231与乳腺正常细胞系MCF10A(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分析，检测EYFP与EBFP2荧光强度。归一化报告基因表达水平＝(实验组EYFP荧光强度/实验组EBFP2荧光强度)/(对照组EYFP荧光强度/对照组EBFP2荧光强度)。The 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).
用CEA368-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用CXCR4-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用hMUC1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用Survivin1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。Use the 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.
归一化报告基因表达水平见图6。所测试癌特异启动子中，CEA205与CXCR4在乳腺癌细胞系MCF7与MDA-MB-231中表达较高而在乳腺正常细胞系MCF10A中表达较低。结果表明：癌特异启动子可以特异性地在乳腺癌细胞系中启动Gal4VP16的表达并激活UAS下游的报告基因。CEA205与CXCR4有较好的表达强度与特异性。The normalized reporter gene expression level is shown in Figure 6. Among the tested cancer-specific promoters, 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.
实验二Experiment two
测定CEA205、CEA368、CXCR4、hMUC1与Survivin1启动5×UAS或4×UAS开关线路的翻转能力。Measure CEA205, CEA368, CXCR4, hMUC1 and Survivin1 to start 5×UAS or 4×UAS switch circuit flipping ability.
将CEA205-Gal4VP16质粒、5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒、5×UAS-LacO-TetRKrab-FF5质粒、CAG-EYFP质粒、pDT7004质粒共转染乳腺癌细胞系MCF7与乳腺正常细胞系MCF10A(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分析，检测EYFP与EBFP2荧光强度，表示无输入信号时开关线路的表达水平。报告基因表达水平＝EBFP2荧光强度/EYFP荧光强度。The CEA205-Gal4VP16 plasmid, 5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 5×UAS-LacO-TetRKrab-FF5 plasmid, CAG-EYFP plasmid, and pDT7004 plasmid were co-transfected into breast cancer cell lines MCF7 and normal breast cell line MCF10A (100ng of each plasmid transfected in each well) were analyzed by flow cytometry 48 hours after transfection to detect the fluorescence intensity of EYFP and EBFP2, which indicates the expression level of the switch circuit when there is no input signal. Reporter gene expression level = EBFP2 fluorescence intensity/EYFP fluorescence intensity.
用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤，表示一侧输入信号时开关线路的表达水平；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤，表示另一侧输入信号时开关线路的表达水平。Use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps, to indicate the expression level of the switch circuit when one side of the signal is input; use U6-shRNA-FF5-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps, Indicates the expression level of the switch circuit when the signal is input from the other side.
用4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4×UAS-LacO-TetRKrab-FF5 plasmid instead After 5×UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps; use U6-shRNA-FF5-CMV-iRFP plasmid instead of pDT7004 plasmid, Perform the above steps.
用CEA368-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CEA368-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CEA368-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 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.
用CXCR4-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CXCR4-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CXCR4-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use CXCR4-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.
用hMUC1-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the hMUC1-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, perform the above steps; use the U6-shRNA-FF5-CMV-iRFP plasmid instead of the pDT7004 plasmid, Perform the above steps.
用hMUC1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use hMUC1-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.
用Survivin1-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with Survivin1-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用Survivin1-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5× UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use Survivin1-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.
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CAG-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 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.
报告基因表达水平见图7。在乳腺正常细胞系MCF10A中所测试的启动子翻转5×UAS开关线路表达量较低，而翻转4×UAS开关线路有一定表达。在乳腺癌细胞系MCF7中所测试的启动未能表现中对5×UAS与4×UAS开关线路较好的翻转能力。结果表明：所测试的启动子在乳腺癌细胞系MCF7中对开关线路的翻转能力不强。The expression level of the reporter gene is shown in Figure 7. In the normal breast cell line MCF10A, 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.
实验三Experiment Three
测定癌特异性microRNA mir-205-5p、mir-141-3p与mir-21在乳腺癌细胞系MCF7、MDA-MB-231与乳腺正常细胞系MCF10A中对各靶位点的抑制强度。The inhibition intensity of cancer-specific microRNAs mir-205-5p, mir-141-3p and mir-21 on each target site in breast cancer cell lines MCF7, MDA-MB-231 and normal breast cell line MCF10A was determined.
将CMV-EYFP-T205 5p x4质粒、CMV-EBFP2质粒、pDT7004质粒共转染乳腺癌细胞系MCF7、MDA-MB-231与乳腺正常细胞系MCF10A(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分析，检测EYFP与EBFP2荧光强度。设置用CMV-EYFP质粒代替CMV-EYFP-T205 5p x4质粒的对照处理。归一化报告基因表达水平＝(实验组EBFP2荧光强度/实验组EYFP荧光强度)/(对照组EBFP2荧光强度/对照组EYFP荧光强度)。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. Set up a control treatment with 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).
用CMV-EYFP-T141 3p x4质粒代替CMV-EYFP-T205 5p x4质粒后，进行上述步骤。用CMV-EYFP-T21x4质粒代替CMV-EYFP-T143 3p x4质粒后，进行上述步骤。After replacing the CMV-EYFP-T205 5p x4 plasmid with the CMV-EYFP-T141 3p x4 plasmid, perform the above steps. After replacing the CMV-EYFP-T143 3p x4 plasmid with the CMV-EYFP-T21x4 plasmid, perform the above steps.
归一化报告基因表达水平见图8。mir-205-5p可以通过靶位点特异性地在乳腺正常细胞系MCF10A中抑制报告基因表达，并有很高的抑制强度，与调研结果一致。mir-141-3p虽然在乳腺癌细胞系MDA-MB-231中抑制强度较低，但在MCF7中有一定的抑制强度。mir-21虽然在乳腺癌细胞系MCF7与MDA-MB-231中抑制强度很高，但在乳腺正常细胞系MCF10A中抑制强度也很高。结果表明：癌特异高表达或低表达的microRNA可以通过靶 位点在乳腺癌细胞系MCF7、MDA-MB-231与乳腺正常细胞系MCF10A中抑制报告基因表达，但特异性不够好。The normalized reporter gene expression level is shown in Figure 8. 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. Although 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. Although 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.
实验四Experiment Four
实验一、二、三的基础上，构建使用CEA205/Survivin1启动子、mir-21与mir-141-3p调控的5×UAS开关线路控制的溶瘤腺病毒，测试对乳腺癌细胞系MCF7、MDA-MB-231、乳腺正常细胞系MCF10A、肝正常细胞系Chang与L02的杀伤能力。On the basis of experiments one, two and three, 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.
在96孔板中每孔接种数量为1e4的细胞。接种24小时后分别加入感染复数为1、10、20、50、100、200、500的B1病毒，设置不加入病毒的空白对照。病毒侵染6天后使用MTS法检测细胞存活率。细胞存活率＝实验组MTS检测值/对照组MTS检测值。Inoculate 1e4 cells per well in a 96-well plate. After 24 hours of inoculation, B1 viruses with a multiplicity of infection of 1, 10, 20, 50, 100, 200, 500 were added respectively, 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.
使用B2病毒代替B1病毒，进行上述步骤。使用B3病毒代替B1病毒，进行上述步骤。Use B2 virus instead of B1 virus to perform the above steps. Use B3 virus instead of B1 virus to perform the above steps.
使用B4病毒代替B1病毒，进行上述步骤。Use B4 virus instead of B1 virus to perform the above steps.
细胞存活率见图9A～9D。四种腺病毒均未能有效杀伤乳腺癌细胞系MCF7与MDA-MB-231而对乳腺正常细胞系MCF10A有一定杀伤作用。结果表明：使用癌特异启动子CEA205/Survivin1与癌特异microRNA mir-21及mir-141-3p调控的开关线路控制的溶瘤腺病毒未能特异性地杀伤乳腺癌细胞系MCF7与MDA-MB-231。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.
实施例3靶向胰腺癌的溶瘤腺病毒的构建与功能验证Example 3 Construction and functional verification of oncolytic adenovirus targeting pancreatic cancer
实验一experiment one
结合实施例1实验结果，测定癌特异性启动子hMUC1在胰腺癌细胞系PANC1与胰腺正常细胞系HTERT-HPNE中的表达强度与特异性。Combined with the experimental results of Example 1, 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.
将hMUC1-Gal4VP16的质粒、UAS-EYFP质粒、CMV-EBFP2质粒共转染胰腺癌细胞系PANC1与胰腺正常细胞系HTERT-HPNE(每孔转染各质粒各100ng)，转染48小时后进行流式细胞术分析，检测EYFP与EBFP2荧光强度。归一化报告基因表达水平＝(实验组EYFP荧光强度/实验组EBFP2荧光强度)/(对照组EYFP荧光强度/对照组EBFP2荧光强度)。The hMUC1-Gal4VP16 plasmid, UAS-EYFP plasmid, and CMV-EBFP2 plasmid were co-transfected into pancreatic cancer cell line PANC1 and pancreatic normal cell line HTERT-HPNE (100ng each plasmid was transfected in each well), and flowed 48 hours after transfection. Cytometry analysis 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).
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒，进行上述步骤。Use CAG-Gal4VP16 plasmid instead of CEA205-Gal4VP16 plasmid to perform the above steps.
归一化报告基因表达水平见图10。hMUC1在胰腺癌细胞系PANC1中表达较高而在胰腺正常细胞系HTERT-HPNE中表达较低。结果表明：癌特异启动子hMUC1可以特异性地在乳腺癌细胞系中启动Gal4VP16的表达并激活UAS下游的报告基因。The normalized reporter gene expression level is shown in Figure 10. 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.
实验二Experiment two
测定hMUC1启动5×UAS或4×UAS开关线路的翻转能力。Determine the flipping capability of hMUC1 to activate the 5×UAS or 4×UAS switch circuit.
将hMUC1-Gal4VP16质粒、5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒、5×UAS-LacO-TetRKrab-FF5质粒、CAG-EYFP质粒、pDT7004质粒共转染胰腺癌细胞系PANC1与胰腺正常细胞系HTERT-HPNE(每孔转染各质粒各100ng)，转染48小时后进行流式细 胞术分析，检测EYFP与EBFP2荧光强度，表示无输入信号时开关线路的表达水平。报告基因表达水平＝EBFP2荧光强度/EYFP荧光强度。The hMUC1-Gal4VP16 plasmid, 5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 5×UAS-LacO-TetRKrab-FF5 plasmid, CAG-EYFP plasmid, and pDT7004 plasmid were co-transfected into pancreatic cancer cell lines PANC1 and normal pancreatic cell line HTERT-HPNE (100ng of each plasmid transfected in each well) were analyzed by flow cytometry 48 hours after transfection to detect the fluorescence intensity of EYFP and EBFP2, which indicates the expression level of the switch circuit when there is no input signal. Reporter gene expression level = EBFP2 fluorescence intensity/EYFP fluorescence intensity.
用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤，表示一侧输入信号时开关线路的表达水平；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤，表示另一侧输入信号时开关线路的表达水平。Use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps, to indicate the expression level of the switch circuit when one side of the signal is input; use U6-shRNA-FF5-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps, Indicates the expression level of the switch circuit when the signal is input from the other side.
用4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid instead of 5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4 plasmid, 4×UAS-LacO-TetRKrab-FF5 plasmid instead After 5×UAS-LacO-TetRKrab-FF5 plasmid, perform the above steps; use U6-shRNA-FF4-CMV-iRFP plasmid instead of pDT7004 plasmid, perform the above steps; use U6-shRNA-FF5-CMV-iRFP plasmid instead of pDT7004 plasmid, Perform the above steps.
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。After replacing the CEA205-Gal4VP16 plasmid with the CAG-Gal4VP16 plasmid, perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF4-CMV-iRFP plasmid, and perform the above steps; replace the pDT7004 plasmid with the U6-shRNA-FF5-CMV-iRFP plasmid, Perform the above steps.
用CAG-Gal4VP16质粒代替CEA205-Gal4VP16质粒，4×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒代替5×UAS-TetO-E1A-P2A-EBFP2-2A-LacI-FF4质粒，4×UAS-LacO-TetRKrab-FF5质粒代替5×UAS-LacO-TetRKrab-FF5质粒后，进行上述步骤；用U6-shRNA-FF4-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤；用U6-shRNA-FF5-CMV-iRFP质粒代替pDT7004质粒，进行上述步骤。Use 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.
报告基因表达水平见图11。在胰腺癌细胞系PANC1中hMUC1可以翻转5×UAS与4×UAS开关线路，但在胰腺正常细胞系HTERT-HPNE中也有一定表达。结果表明：hMUC1在胰腺癌细胞系PANC1中对开关线路有一定翻转能力，但在胰腺正常细胞系HTERT-HPNE中有一定泄漏。The expression level of the reporter gene is shown in Figure 11. In the pancreatic cancer cell line PANC1, 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.
实验三Experiment Three
实验一、二基础上，构建用hMUC1启动子、mir-21与mir-199a-3p调控的5×UAS与4×UAS开关线路控制的溶瘤腺病毒，测试对胰腺癌细胞系PANC1、肝正常细胞系Chang与L02的杀伤能力。On the basis of experiment one and two, construct an oncolytic adenovirus controlled by the 5×UAS and 4×UAS switch circuits regulated by hMUC1 promoter, mir-21 and mir-199a-3p, and test the pancreatic cancer cell line PANC1 and normal liver The killing ability of cell lines Chang and L02.
在96孔板中每孔接种数量为1e4的细胞。接种24小时后分别加入感染复数为0.1、1、5、10、20、50、100的C1病毒，设置不加入病毒的空白对照。病毒侵染6天后使用MTS法检测细胞存活率。细胞存活率＝实验组MTS检测值/对照组MTS检测值。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.
使用C2病毒代替C1病毒，进行上述步骤。Use C2 virus instead of C1 virus to perform the above steps.
细胞存活率见图12。两种腺病毒均能有效地特异性地杀伤胰腺癌细胞系PANC1。C1 在感染复数约20～50间时就表现出明显的杀伤特异性，而C2杀伤效果略弱于pAD043。结果表明：使用癌特异启动子hMUC1与癌特异microRNA mir-21、mir-199a-3p调控的开关线路控制的溶瘤腺病毒可以特异性地杀伤胰腺癌细胞系PANC1The 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
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structure, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above-mentioned terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the characteristics of the different embodiments or examples described in this specification without contradicting each other.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the foregoing within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions and modifications.

Claims (12)

  1. 一种表达系统，其特征在于，包括:An expression system, characterized in that it comprises:
    第一核酸分子，所述第一核酸分子含有细胞特异性启动子；A first nucleic acid molecule, the first nucleic acid molecule containing a cell-specific promoter;
    第二核酸分子，所述第二核酸分子与所述第一核酸分子可操作地连接，所述第二核酸分子编码转录激活因子；A second nucleic acid molecule, the second nucleic acid molecule is operably linked to the first nucleic acid molecule, and the second nucleic acid molecule encodes a transcription activator;
    第三核酸分子，所述第三核酸分子含有所述转录激活因子的第一识别序列；A third nucleic acid molecule, the third nucleic acid molecule containing the first recognition sequence of the transcription activator;
    第四核酸分子，所述第四核酸分子与所述第三核酸分子可操作地连接，所述第四核酸分子含有第一启动子和第一调控元件；A fourth nucleic acid molecule, the fourth nucleic acid molecule is operably linked to the third nucleic acid molecule, and the fourth nucleic acid molecule contains a first promoter and a first regulatory element;
    第五核酸分子，所述第五核酸分子与第四核酸分子可操作地连接，所述第五核酸分子编码第一调控蛋白和目的蛋白，并且所述目的蛋白包括选自病毒复制包装蛋白、免疫效应因子的至少之一；A fifth nucleic acid molecule, the fifth nucleic acid molecule is operably connected to a fourth nucleic acid molecule, the fifth nucleic acid molecule encodes a first regulatory protein and a target protein, and the target protein includes a package protein selected from viral replication packaging proteins, immune At least one of the effect factors;
    第六核酸分子，所述第六核酸分子含有所述转录激活因子的第二识别序列；A sixth nucleic acid molecule, the sixth nucleic acid molecule containing the second recognition sequence of the transcription activator;
    第七核酸分子，所述第七核酸分子与所述第六核酸分子可操作地连接，所述第七核酸分子含有第二启动子和第二调控元件；A seventh nucleic acid molecule, the seventh nucleic acid molecule is operably linked to the sixth nucleic acid molecule, and the seventh nucleic acid molecule contains a second promoter and a second regulatory element;
    第八核酸分子，所述第八核酸分子与第七核酸分子可操作地连接，并且所述第八核酸分子编码第二调控蛋白；An eighth nucleic acid molecule, the eighth nucleic acid molecule is operably linked to a seventh nucleic acid molecule, and the eighth nucleic acid molecule encodes a second regulatory protein;
    第九核酸分子，所述第九核酸分子与所述第五核酸分子可操作地连接，所述第九核酸分子被配置为条件性抑制所述第一调控蛋白的表达；以及A ninth nucleic acid molecule, the ninth nucleic acid molecule is operably linked to the fifth nucleic acid molecule, and the ninth nucleic acid molecule is configured to conditionally inhibit the expression of the first regulatory protein; and
    第十核酸分子，所述第十核酸分子与所述第八核酸分子可操作地连接，所述第十核酸分子被配置为条件性抑制所述第二调控蛋白的表达，A tenth nucleic acid molecule, the tenth nucleic acid molecule is operably linked to the eighth nucleic acid molecule, and the tenth nucleic acid molecule is configured to conditionally inhibit the expression of the second regulatory protein,
    其中，among them,
    所述第一调控元件适于通过结合所述第二调控蛋白抑制所述第一启动子的功能，所述第二调控元件适于通过结合所述第一调控蛋白抑制所述第二启动子的功能；The first regulatory element is adapted to inhibit the function of the first promoter by binding to the second regulatory protein, and the second regulatory element is adapted to inhibit the function of the second promoter by binding to the first regulatory protein. Features;
    所述第九核酸分子与所述第十核酸分子分别独立地借助RNA干扰抑制所述第一调控蛋白或所述第二调控蛋白的表达；The ninth nucleic acid molecule and the tenth nucleic acid molecule independently inhibit the expression of the first regulatory protein or the second regulatory protein by means of RNA interference;
    所述第九核酸分子含有被第一microRNA特异性识别的核酸序列，所述第十核酸分子含有被第二microRNA特异性识别的核酸序列，所述第一microRNA为正常细胞特异性microRNA，所述第二microRNA为异常细胞特异性microRNA；The ninth nucleic acid molecule contains a nucleic acid sequence specifically recognized by a first microRNA, and the tenth nucleic acid molecule contains a nucleic acid sequence specifically recognized by a second microRNA. The first microRNA is a normal cell-specific microRNA. The second microRNA is an abnormal cell specific microRNA;
    所述细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或The cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, and the second microRNA is mir-21; or
    所述细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或The cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, and the second microRNA is mir-21; or
    所述细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或The cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, and the second microRNA is mir-21; or
    所述细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21；或The cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, and the second microRNA is mir-21; or
    所述细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21；或The cell-specific promoter is CEA368, the first microRNA is mir-143-3p, and the second microRNA is mir-21; or
    所述细胞特异性启动子为hMuc1，所述第一microRNA为mir-199a-3p所述第二microRNA为mir-21。The cell-specific promoter is hMuc1, the first microRNA is mir-199a-3p, and the second microRNA is mir-21.
  2. 根据权利要求1所述的表达系统，其特征在于，所述第一识别序列与所述第二识别序列分别独立地选自UAS，tetO以及dCas9的靶标序列至少之一；The expression system of claim 1, wherein the first recognition sequence and the second recognition sequence are independently selected from at least one of UAS, tetO and dCas9 target sequences;
    优选地，所述第一识别序列与所述第二识别序列为5×UAS；Preferably, the first identification sequence and the second identification sequence are 5×UAS;
    任选地，所述转录激活因子为选自Gal4VP16、Gal4vp64、dCas9-VPR、dCas9-VP64、dCas9-VP16、dCas9-VTR以及rtTA至少之一；Optionally, the transcription activator is at least one selected from Gal4VP16, Gal4vp64, dCas9-VPR, dCas9-VP64, dCas9-VP16, dCas9-VTR and rtTA;
    任选地，所述第一启动子与所述第二启动子分别独立地选自miniCMV、TATA box；Optionally, the first promoter and the second promoter are independently selected from miniCMV and TATA box;
    任选地，所述第一调控蛋白和第二调控蛋白分别独立地选自Lacl、tetR、zinc finger、TALE、KRAB、tetR-KRAB、TALE-KRAB、dCas9-KRAB、miniCas9-KRAB、分割dCas9-KRAB至少之一；Optionally, the first regulatory protein and the second regulatory protein are independently selected from Lacl, tetR, zinc finger, TALE, KRAB, tetR-KRAB, TALE-KRAB, dCas9-KRAB, miniCas9-KRAB, split dCas9- At least one of KRAB;
    任选地，所述第一调控元件和所述第二调控元件分别独立地选自tetO、LacO、锌指蛋白靶标序列、TALE蛋白靶标序列、dCas9的靶标序列、miniCas9的靶标序列至少之一；Optionally, the first regulatory element and the second regulatory element are independently selected from at least one of tetO, LacO, zinc finger protein target sequence, TALE protein target sequence, dCas9 target sequence, and miniCas9 target sequence;
    任选地，所述第一调控蛋白是LacI，所述第二调控元件包括多个重复的LacO序列，所述多个重复的LacO序列的至少之一设置在所述第二启动子的下游；Optionally, 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 arranged downstream of the second promoter;
    任选地，所述第二调控蛋白是tetR-KRAB所述第一调控元件包括多个重复的tetO序列，所述多个重复的tetO序列的至少之一设置在所述第一启动子的下游；Optionally, 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 arranged downstream of the first promoter ；
    任选地，所述病毒复制包装相关蛋白包括选自腺病毒E1基因、E1A基因、E1B基因、E2基因和E4基因至少之一；Optionally, 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;
    任选地，所述免疫效应因子包括选自拮抗PD-1基因的抑制序列、拮抗PD-L1基因的抑制序列、拮抗CTLA4基因的抑制序列、拮抗Tim-3基因的抑制序列、GM-CSF、IL-2、IL-12、IL-15至少之一；Optionally, the immune effector includes an inhibitory sequence that antagonizes the PD-1 gene, an inhibitory sequence that antagonizes the PD-L1 gene, an inhibitory sequence that antagonizes the CTLA4 gene, an inhibitory sequence that antagonizes the Tim-3 gene, GM-CSF, At least one of IL-2, IL-12, IL-15;
    任选地，所述目的蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目的蛋白与所述第一调控蛋白之间通过可切割的连接肽连接的；Optionally, 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;
    任选地，所述第一核酸分子与所述第二核酸分子负载在第一表达载体上，所述第三核酸分子、所述第四核酸分子、所述第五核酸分子以及所述第九核酸分子负载在第二表达载体上，所述第六核酸分子、所述第七核酸分子、所述第八核酸分子以及所述第十核酸分子负载在第三表达载体上；Optionally, the first nucleic acid molecule and the second nucleic acid molecule are carried on a first expression vector, and the third nucleic acid molecule, the fourth nucleic acid molecule, the fifth nucleic acid molecule, and the ninth nucleic acid molecule The nucleic acid molecule is loaded on a second expression vector, and 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;
    任选地，所述第一表达载体、第二表达载体和第三表达载体分别独立地选自下列的至少之一：Optionally, the first expression vector, the second expression vector and the third expression vector are each independently selected from at least one of the following:
    质粒、病毒、纳米材料、脂质体、分子耦联载体、裸露DNA、染色体载体、多聚物；Plasmids, viruses, nanomaterials, liposomes, molecular coupling vectors, naked DNA, chromosomal vectors, polymers;
    任选地，所述病毒包括选自腺病毒、牛痘病毒、疱疹病毒、逆转录病毒的至少之一；Optionally, the virus includes at least one selected from the group consisting of adenovirus, vaccinia virus, herpes virus, and retrovirus;
    任选地，所述第一表达载体、第二表达载体和第三表达载体负载在同一个载体上；Optionally, the first expression vector, the second expression vector and the third expression vector are carried on the same vector;
    任选地，所述同一个载体为腺病毒载体。Optionally, the same vector is an adenovirus vector.
  3. 根据权利要求1所述的表达系统，其特征在于，所述细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The expression system of claim 1, wherein the cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first microRNA is mir-21. The recognition sequence and the second recognition sequence are 5×UAS; or
    所述细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS; or
    所述细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS; or
    所述细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS; or
    所述细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The cell-specific promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS; or
    所述细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS; or
    所述细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS; or
    所述细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS; or
    所述细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS; or
    所述细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The cell-specific promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS; or
    所述细胞特异性启动子为hMuc1，所述第一microRNA为mir-199a-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS。The cell-specific promoter is hMuc1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS.
  4. 根据权利要求3所述的表达系统，其特征在于，所述腺病毒载体携带具有SEQ ID NO：1～26任一所示核苷酸序列的核酸；The expression system according to claim 3, wherein the adenovirus vector carries a nucleic acid having a nucleotide sequence shown in any one of SEQ ID NO: 1 to 26;
    任选地，所述腺病毒是通过如下方式获得的：Optionally, the adenovirus is obtained by:
    所述的腺病毒载体去除了与腺病毒复制包装相关的E1基因和部分E3基因，E1A基因通过逐级Golden Gate的方法构建到基因线路中，最终通过Gateway或Gibson的方式将基因线路插入到腺病毒载体中；The adenovirus vector removes the E1 gene and part of the E3 gene related to adenovirus replication and packaging. The E1A gene is constructed into the gene circuit by the step-by-step Golden Gate method, and finally the gene circuit is inserted into the gland by the Gateway or Gibson method. Virus vector
    任选地，所述腺病毒载体为去除E1基因与部分E3基因的B、C亚型腺病毒；Optionally, the adenovirus vector is an adenovirus of subtypes B and C with E1 gene and part of E3 gene removed;
    任选地，所述腺病毒载体为去除E1基因与部分E3基因的5、11、12、34或35型腺病毒；Optionally, 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;
    任选地，SEQ ID NO:1～26任一所示的核苷酸序列在所述5型腺病毒载体的插入位点为E1基因区域、E3基因区域或E4基因区域。Optionally, the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 26 at the insertion site of the type 5 adenovirus vector is an E1 gene region, an E3 gene region or an E4 gene region.
  5. 一种重组病毒，其特征在于，包括：A recombinant virus, characterized in that it comprises:
    第一核酸分子，所述第一核酸分子含有肿瘤细胞特异性启动子；A first nucleic acid molecule, the first nucleic acid molecule containing a tumor cell-specific promoter;
    第二核酸分子，所述第二核酸分子与所述第一核酸分子可操作地连接，所述第二核酸分子编码转录激活因子，所述转录激活因子为Gal4VP16；A second nucleic acid molecule, the second nucleic acid molecule is operably linked to the first nucleic acid molecule, the second nucleic acid molecule encodes a transcription activator, and the transcription activator is Gal4VP16;
    第三核酸分子，所述第三核酸分子含有所述转录激活因子的第一识别序列；A third nucleic acid molecule, the third nucleic acid molecule containing the first recognition sequence of the transcription activator;
    第四核酸分子，所述第四核酸分子与所述第三核酸分子可操作地连接，所述第四核酸分子含有第一启动子和第一调控元件，所述第一启动子为miniCMV，所述第一调控元件包括多个重复的tetO序列，所述多个重复的tetO序列的至少之一设置在所述第一启动子的下游；A fourth nucleic acid molecule, said fourth nucleic acid molecule is operably linked to said third nucleic acid molecule, said fourth nucleic acid molecule contains a first promoter and a first regulatory element, said first promoter is miniCMV, and The first regulatory element includes a plurality of repeated tetO sequences, and at least one of the plurality of repeated tetO sequences is arranged downstream of the first promoter;
    第五核酸分子，所述第五核酸分子与第四核酸分子可操作地连接，所述第五核酸分子编码第一调控蛋白，所述第一调控蛋白为LacI；A fifth nucleic acid molecule, the fifth nucleic acid molecule is operably connected to a fourth nucleic acid molecule, the fifth nucleic acid molecule encodes a first regulatory protein, and the first regulatory protein is LacI;
    所述第五核酸分子进一步包括编码目的蛋白的序列，并且所述目的蛋白包括病毒复制蛋白、免疫效应因子，所述免疫效应因子是以单独或融合蛋白的形式表达的，并且所述毒复制蛋白和所述效应因子之间通过可切割的连接肽连接的，The fifth nucleic acid molecule further includes a sequence encoding a target protein, and the target protein includes a viral replication protein, an immune effector, and the immune effector is expressed in the form of a single or a fusion protein, and the viral replication protein And the effector is connected by a cleavable linking peptide,
    所述目的蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目的蛋白与所述第一调控蛋白之间通过可切割的连接肽连接的；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;
    第六核酸分子，所述第六核酸分子含有所述转录激活因子的第二识别序列；A sixth nucleic acid molecule, the sixth nucleic acid molecule containing the second recognition sequence of the transcription activator;
    第七核酸分子，所述第七核酸分子与所述第六核酸分子可操作地连接，所述第七核酸分子含有第二启动子和第二调控元件，所述第二启动子为miniCMV，所述第二调控元件包 括多个重复的LacO序列，所述多个重复的LacO序列的至少之一设置在所述第二启动子的下游；A seventh nucleic acid molecule, the seventh nucleic acid molecule is operably linked to the sixth nucleic acid molecule, the seventh nucleic acid molecule contains a second promoter and a second regulatory element, the second promoter is miniCMV, and The second regulatory element includes a plurality of repeated LacO sequences, and at least one of the plurality of repeated LacO sequences is arranged downstream of the second promoter;
    第八核酸分子，所述第八核酸分子与第七核酸分子可操作地连接，并且所述第八核酸分子编码第二调控蛋白，所述第二调控蛋白为tetR-KRAB；An eighth nucleic acid molecule, the eighth nucleic acid molecule is operably linked to a seventh nucleic acid molecule, and the eighth nucleic acid molecule encodes a second regulatory protein, and the second regulatory protein is tetR-KRAB;
    第九核酸分子，所述第九核酸分子与所述第五核酸分子可操作地连接，所述第九核酸分子被配置为条件性抑制所述第一调控蛋白的表达，所述第九核酸分子含有被第一microRNA特异性识别的核酸序列，所述第一microRNA为正常细胞特异性microRNA；以及A ninth nucleic acid molecule, the ninth nucleic acid molecule is operably linked to the fifth nucleic acid molecule, the ninth nucleic acid molecule is configured to conditionally inhibit the expression of the first regulatory protein, the ninth nucleic acid molecule Contains a nucleic acid sequence specifically recognized by a first microRNA, the first microRNA being a normal cell specific microRNA; and
    第十核酸分子，所述第十核酸分子与所述第八核酸分子可操作地连接，所述第十核酸分子被配置为条件性抑制所述第二调控蛋白的表达，所述第十核酸分子含有被第二microRNA特异性识别的核酸序列，所述第二microRNA为肿瘤细胞特异性microRNA，A tenth nucleic acid molecule, the tenth nucleic acid molecule is operably linked to the eighth nucleic acid molecule, the tenth nucleic acid molecule is configured to conditionally inhibit the expression of the second regulatory protein, the tenth nucleic acid molecule Contains a nucleic acid sequence specifically recognized by a second microRNA, the second microRNA being a tumor cell specific microRNA,
    其中，among them,
    所述第一调控元件适于通过结合所述第二调控蛋白抑制所述第一启动子的功能，所述第二调控元件适于通过结合所述第一调控蛋白抑制所述第二启动子的功能；The first regulatory element is adapted to inhibit the function of the first promoter by binding to the second regulatory protein, and the second regulatory element is adapted to inhibit the function of the second promoter by binding to the first regulatory protein. Features;
    所述肿瘤细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The tumor cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS ;or
    所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The tumor cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS ;or
    所述肿瘤细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The tumor cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS ;or
    所述肿瘤细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The tumor cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS ;or
    所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为5×UAS；或The tumor cell-specific promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 5×UAS ;or
    所述肿瘤细胞特异性启动子为CEA205，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The tumor cell-specific promoter is CEA205, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS ;or
    所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The tumor cell-specific promoter is CEA368, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS ;or
    所述肿瘤细胞特异性启动子为CXCR4，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The tumor cell-specific promoter is CXCR4, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS ;or
    所述肿瘤细胞特异性启动子为Survivin1，所述第一microRNA为mir-135a-5p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The tumor cell-specific promoter is Survivin1, the first microRNA is mir-135a-5p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS ;or
    所述肿瘤细胞特异性启动子为CEA368，所述第一microRNA为mir-143-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS；或The tumor cell-specific promoter is CEA368, the first microRNA is mir-143-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS ;or
    所述肿瘤细胞特异性启动子为hMuc1，所述第一microRNA为mir-199a-3p，所述第二microRNA为mir-21，所述第一识别序列与所述第二识别序列为4×UAS。The tumor cell-specific promoter is hMuc1, the first microRNA is mir-199a-3p, the second microRNA is mir-21, and the first recognition sequence and the second recognition sequence are 4×UAS .
  6. 根据权利要求5所述的重组病毒，其特征在于，所述重组病毒为选自腺病毒、牛痘病毒、逆转录病毒、疱疹病毒的至少之一；The recombinant virus according to claim 5, wherein the recombinant virus is at least one selected from the group consisting of adenovirus, vaccinia virus, retrovirus, and herpes virus;
    优选地，所述重组病毒为腺病毒；Preferably, the recombinant virus is an adenovirus;
    任选地，所述免疫效应因子包括选自拮抗PD-1基因的抑制序列、拮抗PD-L1基因的抑制序列、拮抗CTLA4基因的抑制序列、拮抗Tim-3基因的抑制序列、IL-2、IL-12、IL-15、GM-CSF至少之一。Optionally, the immune effector includes an inhibitory sequence that antagonizes the PD-1 gene, an inhibitory sequence that antagonizes the PD-L1 gene, an inhibitory sequence that antagonizes the CTLA4 gene, an inhibitory sequence that antagonizes the Tim-3 gene, IL-2, At least one of IL-12, IL-15, GM-CSF.
  7. 一种重组细胞，其特征在于，含有权利要求1～4任一项所述的表达系统。A recombinant cell characterized by containing the expression system according to any one of claims 1 to 4.
  8. 根据权利要求7所述的重组细胞，其特征在于，所述表达系统的至少一部分整合于所述重组细胞的基因组中。The recombinant cell according to claim 7, wherein at least a part of the expression system is integrated into the genome of the recombinant cell.
  9. 权利要求1～4任一项所述的表达系统、权利要求5～6任一项所述的重组病毒、权利要求7～8任一项所述的重组细胞在制备药物中的用途，所述药物用于治疗胃癌；The use of the expression system according to any one of claims 1 to 4, the recombinant virus according to any one of claims 5 to 6, and the recombinant cell according to any one of claims 7 to 8 in the preparation of medicines, Drugs used to treat gastric cancer;
    任选地，所述药物用于治疗胰腺癌。Optionally, the medicament is used to treat pancreatic cancer.
  10. 一种药物组合物，其特征在于，包含权利要求5～6任一项所述的重组病毒或权利要求7～8任一项所述的重组细胞。A pharmaceutical composition characterized by comprising the recombinant virus according to any one of claims 5 to 6 or the recombinant cell according to any one of claims 7 to 8.
  11. 根据权利要求10所述的药物组合物，其特征在于，进一步包括药学上可接受的辅料；The pharmaceutical composition of claim 10, further comprising pharmaceutically acceptable excipients;
    任选地，进一步包括其他治疗胃癌的药物；Optionally, it further includes other drugs for treating gastric cancer;
    任选地，所述其他治疗胃癌的药物包括选自Pembrolizumab、Ogivri、Everolimus、Lanreotide、Ramucirumab、Apatinib、Trastuzumab的至少之一；Optionally, the other drugs for treating gastric cancer include at least one selected from Pembrolizumab, Ogivri, Everolimus, Lanreotide, Ramucirumab, Apatinib, and Trastuzumab;
    任选地，进一步包括其他治疗胰腺癌的药物；Optionally, it further includes other drugs for the treatment of pancreatic cancer;
    任选地，所述其他治疗胰腺癌的药物包括选自Lanreotide、Abraxane、Olaparib、Afinitor、Erlotinib、Everolimus、5-FU、Gemzar、Sunitinib、Onivyde、Gemzar的至少之一。Optionally, 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.
  12. 一种治疗或预防胃癌或胰腺癌的方法，其特征在于，包括：给与患者治疗有效量的权利要求1～4任一项所述的表达系统、权利要求5～6任一项所述的重组病毒、权利要求7～8任一项所述的重组细胞。A method for treating or preventing gastric cancer or pancreatic cancer, characterized in that it comprises: administering to a patient a therapeutically effective amount of the expression system according to any one of claims 1 to 4, and the expression system according to any one of claims 5 to 6 Recombinant virus, the recombinant cell according to any one of claims 7 to 8.
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