WO2018171103A1 - 可编程的溶瘤病毒疫苗系统及其应用 - Google Patents

可编程的溶瘤病毒疫苗系统及其应用 Download PDF

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WO2018171103A1
WO2018171103A1 PCT/CN2017/096043 CN2017096043W WO2018171103A1 WO 2018171103 A1 WO2018171103 A1 WO 2018171103A1 CN 2017096043 W CN2017096043 W CN 2017096043W WO 2018171103 A1 WO2018171103 A1 WO 2018171103A1
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nucleic acid
acid molecule
protein
promoter
expression
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PCT/CN2017/096043
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English (en)
French (fr)
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谢震
黄慧雅
刘乙齐
廖微曦
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清华大学
北京合生基因科技有限公司
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Priority to US16/470,520 priority Critical patent/US20200017881A1/en
Priority to CN201780002478.XA priority patent/CN108064305B/zh
Priority to KR1020197031190A priority patent/KR102409077B1/ko
Priority to AU2017405929A priority patent/AU2017405929B2/en
Priority to JP2020500941A priority patent/JP6961788B2/ja
Priority to EP17901776.9A priority patent/EP3604548A4/en
Publication of WO2018171103A1 publication Critical patent/WO2018171103A1/zh

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Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to an expression system and its use, and more particularly, to the use of an expression system, a recombinant virus, a recombinant cell, and an expression system, a recombinant virus, and a recombinant cell in the preparation of a medicament, Further, the present invention also relates to a method of expressing a protein of interest using an expression system.
  • 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 the expression of some of the less virulent strains in nature, allowing them to express their packaging in tumor cells and then achieve oncolysis. There are two main principles for using tumorigenic viruses to recognize tumor cells. First, they selectively infect tumor cells by inactivating or defective tumor suppressor genes in target cells. Second, they selectively use tumor-specific promoters to regulate viruses. The expression of key genes allows the oncolytic virus to replicate in large amounts in tumor cells and express toxic proteins to destroy tumor cells, and/or simultaneously secrete cytokines to stimulate the autoimmune system to attack tumor cells.
  • oncolytic virus cannot replicate in normal organism cells without killing effect, so the oncolytic virus has higher anti-tumor effect and lower side effects.
  • oncolytic virus treatment has caused widespread concern, and relevant research has made great progress.
  • adenovirus, herpes simplex virus-1 (HSV-1), Newcastle disease virus, etc. have been transformed into oncolytic viruses.
  • HSV-1 herpes simplex virus-1
  • Newcastle disease virus etc.
  • oncolytic adenovirus products (Gendicine and oncorine) have been used in clinical treatment in China, mainly for the treatment of head and neck cancer, sinus cancer. The principle of Gendicine and oncorine is similar.
  • OncoVEX GM-CSF The genetically engineered herpes simplex virus OncoVEX GM-CSF, developed by biotechnology company BioVex, was approved by the FDA in October 2015 to become the first oncolytic virus product to be marketed in the US and Europe.
  • OncoVex selectively kills tumor cells while expressing an immune response that secretes GM-CSF to initiate the body's immune system to kill residual tumor cells and their metastatic sites.
  • a phase II trial of metastatic melanoma published by BioVex in 2009 showed that 26% of 50 patients responded to treatment and 8 patients returned to full health. The company was acquired by Agmen for $1 billion in 2011 for advancement.
  • Adl ⁇ Ad52 human adenoviruses
  • Ad2A and E1B which are involved in cell transformation
  • E2 region encodes DNA binding.
  • the protein is involved in viral replication; the E3 region encodes a glycoprotein that appears on the surface of the host cell; the E4 region is located at the right end of the Ad2 genome and is regulated by the DNA binding protein encoded by the E2 region; the fifth transcription unit synthesizes the Ad2 protein during the viral infection.
  • the cellular transcription factor first binds to the enhancer upstream of the E1A region, expressing the E1A protein, which regulates cellular metabolism and makes the viral DNA more easily replicated in the cell.
  • the E1A protein also activates promoters of other early genes (E1B, E2A, E2B, E3, and E4), where E2B drives three additional early gene transcriptional terminal protein precursors (pTPs) involved in viral replication.
  • ssDBP single-stranded DNA binding proteins
  • DNA pol DNA polymerase
  • E1A expression to regulate the replication of packaging of adenovirus in targeted cells
  • removal of some viral packaging non-essential genes to reduce virus-to-non- The toxicity of the target cells, while increasing the packaging capacity of the virus, such as E3, E4, etc.
  • replacing the coat protein of the adenovirus changing the targeting of the virus to specific cells and tissues.
  • the present invention aims to solve at least one of the related technical problems to some extent.
  • the inventors designed and constructed a gene line that is suitable for adenovirus regulation and can respond to different microenvironments.
  • this genetic line the inventors utilized multiple levels of biomarkers. Specifically, first, a specific promoter is used as a master switch for regulating gene lines, that is, a specific promoter is used to regulate the expression of the total activator, and the expression of the adenovirus E1A gene is further regulated. Second, the target sequence of the microRNA is used to respond to the microRNA expression characteristics of different microenvironments as a secondary switch for regulating gene lines.
  • the closed two-way suppression switch can respond to changes in the microenvironment more efficiently, while effectively avoiding Give way.
  • the E1B gene was removed, and the ability of E1B to recognize the complementary deletion of P53 gene further improved the ability of adenovirus to distinguish tumor cells from normal cells.
  • the removal of the E3 gene reduces the toxic effects of oncolytic adenovirus on normal cells and increases the packaging capacity of adenoviral vectors.
  • the genome length of type 2 and type 5 human adenoviruses is 36K. It is very difficult and time consuming to reconstruct adenovirus using traditional single plasmid construction methods (such as restriction enzyme ligation).
  • the inventors designed a rapid construction method for the adenovirus constructed by the gene line involved in the present invention: the first step is to construct a first-level component library, and the desired components are constructed on the corresponding plasmids.
  • the primary component library includes three libraries: a suppressor element A library mainly expressing a suppressor element on one side of the gene line, an E1A gene of an adenovirus, and an effector gene (such as an immune factor or a killer gene) co-expressed with an adenovirus; and a suppressor element B
  • the library mainly expresses the suppressor gene on the other side of the gene line, regulates the effective flipping of the gene line, and enhances the safety of the switch; the specific promoter library regulates the total switch of the downstream gene line through the tissue or tumor-specific promoter.
  • the third step is to integrate the gene line into the modified adenoviral vector by Gateway (removing the E1 gene to facilitate manual control of the adenovirus; removing part of the E3 sequence to expand the packaging capacity).
  • oncolytic adenoviruses can carry and simultaneously express multiple genes.
  • the inventors simultaneously expressed single or multiple cellular immune-related genes: such as IL-2, GM-CSF, and anti- PD-1 scFv, anti-PD-L1 scFv, and fusion proteins between these factors and the like.
  • immune-related genes such as IL-2, GM-CSF, and anti- PD-1 scFv, anti-PD-L1 scFv, and fusion proteins between these factors and the like.
  • oncolytic viruses Due to the carrying of immune-related genes, oncolytic viruses cause systemic immune responses while attacking tumor cells, but there are also risks and hidden dangers that cause immune overreaction. Therefore, what kind of immune response gene is carried, and the corresponding treatment of how much dose of virus, as well as the mode of administration of the virus treatment, strongly influence the therapeutic effect of the oncolytic virus.
  • the inventors attempted to model the process of infecting adenovirus-infected target cells by means of bioinformatics, and studied the characteristics of oncolytic adenovirus killing tumor cells, and improved the treatment of oncolytic adenovirus as much as possible. effect.
  • the invention proposes an expression system.
  • the expression system comprises: a first nucleic acid molecule comprising a 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 encoding a transcriptional activator; a third nucleic acid molecule comprising a first recognition sequence of the transcriptional activator; a fourth nucleic acid molecule, the fourth nucleic acid molecule
  • the third nucleic acid molecule is operably linked, the fourth nucleic acid molecule comprising a first promoter and a first regulatory element; a fifth nucleic acid molecule, the fifth nucleic acid molecule being operably linked to a fourth nucleic acid molecule,
  • the fifth nucleic acid molecule encodes a first regulatory protein; a sixth nucleic acid molecule comprising a second recognition sequence of the transcriptional activator; a seventh nucleic acid molecule
  • the expression system may further comprise at least one of the following additional technical features:
  • the cell-specific promoter is a tumor cell-specific promoter selected from the group consisting of an alpha-fetoprotein-specific promoter, a Survivin gene promoter, and a human telomerase.
  • Reverse transcriptase gene promoter cholecystokinin A receptor gene promoter, carcinoembryonic antigen promoter, proto-oncogene human epidermal growth factor receptor 2 promoter, prostaglandin internal oxidase reductase 2 promoter, chemokine At least one of receptor-4, E2F-1 gene promoter, mucin promoter, prostate specific antigen, human tyrosinase-related protein 1, and tyrosinase promoter.
  • the expression system of the examples of the present application can be initiated in the microenvironment of specific tumor cells, and the specificity of the expression system for gene expression regulation is further enhanced.
  • the transcriptional activator is at least one selected from the group consisting of Gal4VP16, Gal4VP64, Gal4esn, dCas9-VP16, dCas9-VP64, dCas9-VPR, dCas9-VTR, and rtTA.
  • the first recognition sequence and the second recognition sequence are each independently selected from at least one of a target sequence of 5 ⁇ UAS, 7 ⁇ tetO and dCas9.
  • the first promoter and the second promoter are each independently selected from a miniCMV, a TATA box.
  • the first regulatory protein and the second regulatory protein are each independently selected from at least one of Lacl, tetR, zinc finger (zinc finger), KRAB, tetR-KRAB, dCas9-KRAB.
  • the first regulatory element and the second regulatory element are each independently selected from at least one of a tetO, a LacO, a zinc finger target site, and a target sequence of dCas9.
  • the first regulatory protein is LacI
  • the second regulatory element comprises a plurality of repeating LacO sequences, at least one of the plurality of repeated LacO sequences being set at the second promoter Downstream.
  • the LacO sequence can be specifically bound, thereby inhibiting the function of the second promoter.
  • the LacI/LacO inhibition system of the embodiment of the present invention it has been experimentally shown that the system can effectively inhibit the expression of genes downstream of the promoter.
  • the second regulatory protein is tetR-KRAB
  • the first regulatory element comprises a plurality of repeated tetO sequences, at least one of the plurality of repeated tetO sequences being set at the first initiation Downstream of the child.
  • At least one of the fifth nucleic acid molecule and the ninth nucleic acid molecule further comprises a sequence encoding a protein of interest.
  • the fifth nucleic acid molecule comprises a sequence encoding the protein of interest, and the protein of interest comprises at least one selected from the group consisting of a viral replication packaging protein and an immune effector.
  • the viral replication packaging protein, immune effector may be present 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 the immune effector can effectively activate the body's immune system, thereby promoting immune killing of specific cells such as tumor cells.
  • the virus replication packaging-related protein comprises at least one selected from the group consisting of an adenovirus E1 gene, an E1A gene, an E1B gene, an E2 gene, and an E4 gene.
  • the immune effector comprises a sequence selected from the group consisting of 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, and GM.
  • - at least one of CSF, IL-2, IL-12, IL-15.
  • the above immune effector factors may be present 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 linked by a cleavable linker peptide.
  • the target protein and the first regulatory protein are regulated and expressed under the same promoter, and are expressed after being cleaved at the linker peptide, and 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 ninth nucleic acid molecule and the tenth nucleic acid molecule independently inhibit expression of the first regulatory protein or the second regulatory protein, respectively, by RNA interference.
  • the microRNA is a specific microRNA expressed by different cellular microenvironments
  • the ninth or tenth nucleic acid molecule is a specific target sequence of the microRNA, and then the microRNA target expressed in the specific microenvironment can be realized by RNA interference (RNAi)
  • RNAi RNA interference
  • the ninth nucleic acid molecule comprises a nucleic acid sequence specifically recognized by a first microRNA, the tenth nucleic acid molecule comprising a nucleic acid sequence specifically recognized by a second microRNA, the first microRNA being normal A cell-specific microRNA, the second microRNA being an abnormal cell-specific microRNA.
  • the first regulatory protein is expressed in abnormal cells, and is not expressed or underexpressed in normal cells, while the second regulatory protein is expressed in normal cells and is not expressed or expressed in abnormal cells.
  • the first microRNA comprises at least one selected from the group consisting of miR199a, miR95, miR125, miR125b, Let-7, miR143, miR145 and miR200C.
  • the above microRNA is highly expressed in normal liver cells.
  • the second microRNA comprises at least one selected from the group consisting of miR21, miR223, miR224, miR221, miR18, miR214, miR146a and miR1792.
  • the second microRNA is a microRNA specifically expressed by liver cancer cells (HepG2, Huh7, PLC). Furthermore, the first regulatory protein is expressed in liver cancer cells, and is not expressed or expressed in normal cells, while the second regulatory protein is expressed in normal cells and is not expressed or expressed in liver cancer cells.
  • the first nucleic acid molecule and the second nucleic acid molecule are supported on a first expression vector
  • the selected ninth nucleic acid molecule is supported on a second expression vector
  • the sixth nucleic acid molecule, the seventh nucleic acid molecule, the eighth nucleic acid molecule, and optionally the tenth nucleic acid molecule are loaded in a third On the expression vector.
  • the first, second and third expression vectors serve as a loading vector for the expression system to effect regulation of the specific expression of the gene of interest in a suitable microenvironment, such as a cell.
  • the selection of the expression vector is not particularly limited as long as the function of the expression system in a suitable microenvironment can be achieved.
  • the first expression vector, the second expression vector and the third expression vector are each independently selected from at least one of the following:
  • Plasmids, viruses, stable cell lines, and other material carriers such as nanomaterials, liposomes, molecularly coupled vectors, naked DNA, chromosomal vectors, polymers.
  • the virus comprises at least one selected from the group consisting of an adenovirus, a vaccinia virus, a herpes virus, and a retrovirus.
  • the first expression vector, the second expression vector and the third expression vector are constructed to be loaded on the same vector.
  • the order of joining 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.
  • loading on the same expression vector can effectively solve the problem that the co-transfection efficiency of a plurality of large fragment vectors is extremely low.
  • the same vector is an adenoviral vector.
  • adenovirus As a gene therapy vector, adenovirus has a wide range of main genes, low pathogenicity to humans, infection and expression of genes in proliferating and non-proliferating cells, high titer, homology with human genes, no insertional mutagenicity, and can be cultured in suspension. The advantage of amplifying in the liquid and simultaneously expressing multiple genes.
  • the first expression vector comprises BsaI, AFP III, Gal4VP16 and BsaI (BsaI-AFP III-Gal4VP16-BsaI) from the 5' end to the 3' end.
  • the first expression vector carries a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 1.
  • the second expression vector comprises: BsaI, 5 ⁇ UAS, tetO, miniCMV, tetO, E1A, 2A, immune effector (Effector), LacI, microRNA199a from 5' to 3'.
  • BsaI BsaI-5 ⁇ UAS-tetO-miniCMV-tetO-E1A-2A-Effector-LacI-miR199a target site-BsaI).
  • the effector specifically refers to IL-2, hGM-CSF, mGM-CSF, anti-PD-1 scFv, anti-PD-L1 scFv, IL-2 fusion anti-PD-1 scfv, hGM-CSF fusion anti- PD-1scfv, mGM-CSF fusion anti-PD-1scfv, IL-2 fusion anti-PD-L1scfv, hGM-CSF fusion anti-PD-L1scfv, mGM-CSF fusion anti-PD-L1scfv.
  • the second expression vector carries a nucleic acid having the nucleotide sequence shown in any one of SEQ ID NOS: 2 to 7.
  • SEQ ID NO: 2 is the sequence of BsaI-5 ⁇ UAS-tetO-miniCMV-tetO-E1A-2A-EBFP-2A-LacI-miR199a target site-BsaI;
  • SEQ ID NO: 3 is the sequence of BsaI-5 ⁇ UAS-tetO-miniCMV-tetO-E1A-2A-hIL-2-2A-LacI-miR199a target site-BsaI;
  • SEQ ID NO: 4 is the sequence of BsaI-5 ⁇ UAS-tetO-miniCMV-tetO-E1A-2A-hGM-CSF-2A-LacI-miR199a target site-BsaI;
  • SEQ ID NO: 5 is the sequence of BsaI-5 ⁇ UAS-tetO-miniCMV-tetO-E1A-2A-mGM-CSF-2A-LacI-miR199a target site-BsaI;
  • SEQ ID NO: 6 is the sequence of BsaI-5 ⁇ UAS-tetO-miniCMV-tetO-E1A-2A-anti-PD-1scFv-2A-LacI-miR199a target site-BsaI;
  • SEQ ID NO: 7 is the sequence of BsaI-5 x UAS-tetO-miniCMV-tetO-E1A-2A-anti-PD-L1scFv-2A-LacI-miR199a target site-BsaI.
  • the third expression vector comprises BsaI, 5 ⁇ UAS, LacO, miniCMV, LacO, tetR-KRAB, microRNA21 specific target site and BsaI from 5' to 3'.
  • the third expression vector carries a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 8.
  • the adenovirus comprises: from 5' to 3', inverted terminal repeat ITR, package signal, AFPIII, Gal4VP16, 5 x UAS ,tetO,miniCMV,tetO,E1A,2A,Effector,2A,LacI,miR199a target site,5 ⁇ UAS,LacO,miniCMV,LacO,tetR-KRAB,miR21target site,adenovirus E2 gene region,E3 gene region (removed Region 28922-30801), E4 gene region, second inverted terminal repeat (ITR).
  • the inventors injected the above adenovirus into a tumor mouse model to significantly inhibit the growth of mouse tumors.
  • the above adenovirus can be used as a safe and effective oncolytic virus vaccine to achieve safe and effective specific killing of related tumors.
  • the adenoviral vector carries a nucleic acid having the nucleotide sequence shown in SEQ ID NOS: 9 to 14.
  • SEQ ID NO: 9 is the sequence of the EBFP-bearing nucleic acid carried by the adenoviral vector
  • SEQ ID NO: 10 is the sequence of a nucleic acid carrying hIL-2 carried by an adenoviral vector
  • SEQ ID NO: 11 is the sequence of a nucleic acid carrying hGMCSF carried by an adenoviral vector
  • SEQ ID NO: 12 is the sequence of a nucleic acid carrying mGMCSF carried by an adenoviral vector
  • SEQ ID NO: 13 is the sequence of a nucleic acid carrying an anti-PD-1 scFv carried by an adenoviral vector
  • SEQ ID NO: 14 is the sequence of a nucleic acid carrying an anti-PD-L1 scFv carried by an adenoviral vector.
  • the adenovirus is obtained by removing the E1 gene and a part of the E3 gene associated with adenoviral replication packaging, and the E1A gene is constructed by a stepwise Golden Gate method.
  • the logic is finally inserted into the adenoviral vector by way of Gateway or Gibson.
  • the invention proposes a recombinant virus.
  • the recombinant virus comprises: a first nucleic acid molecule, the first nucleic acid molecule comprising a tumor cell-specific promoter, and the tumor cell-specific promoter is an alpha-fetoprotein-specific promoter; a nucleic acid molecule operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a transcriptional activator, the transcriptional activator is Gal4VP16; a third nucleic acid molecule, the third The nucleic acid molecule comprises a first recognition sequence of the transcriptional activator, the first recognition sequence being 5 x UAS; a fourth nucleic acid molecule operably linked to the third nucleic acid molecule, The fourth nucleic acid molecule comprises a first promoter and a first regulatory element, the first promoter is a miniCMV, and the first regulatory element comprises a plurality of repeating tet
  • the first regulatory protein LacI and the target protein are specifically expressed in tumor cells
  • the second regulatory protein tetR-KRAB is specifically not expressed or underexpressed in tumor cells, and thus the inhibition mechanism of the first promoter miniCMV mediated by tetR-KRAB is released, the first regulatory protein LacI, the target protein in the first promoter miniCMV Under the control of efficacious expression, the LacI-mediated inhibition mechanism effectively inhibited the function of the second promoter miniCMV, and the expression of tetR-KRAB was further inhibited.
  • expression of the protein in the tumor cell can be more specifically expressed (such as the target protein Lacl) or not expressed (such as tetR-KRAB), and the expression efficiency or non-expression efficiency in the tumor cell can be achieved. And high specificity.
  • the above recombinant virus may further include at least one of the following additional technical features:
  • the recombinant virus is at least one selected from the group consisting of a retrovirus, an adenovirus, a herpes virus, and a vaccinia virus.
  • the recombinant virus is an adenovirus.
  • adenovirus has a wide host range as a gene therapy vector, low pathogenicity to humans, infection and expression of genes in proliferating and non-proliferating cells, high titer, homology to human genes, and no insertional mutagenicity. The advantage of being able to amplify in suspension culture and simultaneously express multiple genes.
  • the immune effector comprises a restriction sequence selected from the group consisting of an antagonistic PD-1 gene, an inhibitory sequence which antagonizes the PD-L1 gene, an inhibitory sequence which antagonizes the CTLA4 gene, an inhibitory sequence which antagonizes the Tim-3 gene, and 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 invention proposes a recombinant cell.
  • the recombinant cell comprises an expression system as described above.
  • the recombinant cell according to the embodiment of the invention can effectively activate the systemic immune response of the human body, and attack the xenogeneic cells, such as tumor cells, with high safety and specificity.
  • the above recombinant cell may further comprise at least one of the following additional technical features:
  • At least a portion of the expression system is integrated into the genome of the recombinant cell.
  • the expression system replicates as the recombinant cell genome replicates, and the expression system continues to be effective in regulating the expression of the protein of interest.
  • the invention provides the use of the expression system described above, the recombinant virus described above, and the recombinant cells described above for the preparation of a medicament for the treatment of cancer.
  • the expression system described in the present application can achieve specific expression of a target protein in tumor cells, and the drug according to the embodiment of the present application is more effective, specific, and safer for treating cancer.
  • the above use may further include at least one of the following additional technical features:
  • the cancer comprises liver cancer, lung cancer, colorectal cancer, melanoma, breast cancer or prostate cancer.
  • the inventors have found that the therapeutic effect of the drug according to the embodiment of the present invention on liver cancer, lung cancer, colorectal cancer, melanoma, breast cancer or prostate cancer is more remarkable.
  • the invention proposes a method of expressing a protein of interest using an expression system, which is the expression system described above.
  • the method comprises: (1) causing the fifth nucleic acid molecule to comprise a nucleic acid sequence encoding the protein of interest; (2) inhibiting the tenth nucleic acid molecule from the second regulatory protein Expression to express the protein of interest.
  • the second regulatory protein-mediated inhibition mechanism of the first promoter is released, and the target protein is co-operated by the cell-specific promoter and the first promoter. Highly expressed in specific cells.
  • the above method may further include at least one of the following additional technical features:
  • the expression is carried out in a cell.
  • the cells can provide a microenvironment for the expression of the protein of interest, and the expression of the protein of interest in the cell is more efficient.
  • the tenth nucleic acid molecule comprises a nucleic acid sequence specifically recognized by a second microRNA, and in step (2) comprises contacting the second microRNA with the tenth nucleic acid molecule.
  • the invention proposes a method of expressing a protein of interest using an expression system, which is the expression system described above.
  • the method comprises: (1) causing the eighth nucleic acid molecule to comprise a nucleic acid sequence encoding a first protein of interest, the fifth nucleic acid molecule comprising a nucleic acid sequence encoding a second protein of interest; 2) expressing the first protein of interest or the second protein of interest by one of: inhibiting expression of the first regulatory protein by the ninth nucleic acid molecule to express the first protein of interest; A tenth nucleic acid molecule inhibits expression of the second regulatory protein to express the second protein of interest.
  • the second regulatory protein-mediated inhibition of the first promoter is released, and the second protein of interest is co-operating with the cell-specific promoter and the first promoter.
  • the first protein of interest is efficiently activated in a specific cell by a combination of a cell-specific promoter and a second promoter. expression.
  • fusion protein described herein refers to a protein that is co-transcribed under the control of the same promoter, including a fusion protein that is not linked between proteins, or a fusion protein linked by other linker peptides (such as GGGS or 2A sequence). .
  • Figure 1 is a schematic diagram of the original structure of the adenovirus of the Gateway system
  • FIG. 1 Schematic diagram of the original structure of the Adenovirus of the Golden Gate system
  • Figure 4 is a diagram showing the detection of the hAFP gene in the cell line Chang by the method of real-time quantitative PCR according to an embodiment of the present invention. Expression levels in HepG2, Huh7, Hep3B, PLC and Hepa1-6;
  • Figure 5 is a promoter for engineering a human AFP gene according to an embodiment of the present invention.
  • FIG. 6 is a schematic transmission system of the modified AFP promoter to Chang and HepG2 according to an embodiment of the present invention
  • FIG. 7 is a schematic diagram of a microRNA detection system in accordance with an embodiment of the present invention.
  • Figure 8 is a diagram showing significant differential expression of microRNA markers in different cell lines in accordance with an embodiment of the present invention.
  • FIG. 9 is a schematic diagram of a double suppression switch based on the LacI and tetR-KRAB genes according to an embodiment of the present invention.
  • FIG. 10 is a diagram showing a dual suppression switch in response to a synthetic shRNA signal to achieve efficient flipping in accordance with an embodiment of the present invention
  • 11 is a dual suppression switch in response to various activation factors in accordance with an embodiment of the present invention.
  • CGGA Cluster Golden-Gate and Gateway/Gibson Assemble method
  • Figure 13 is a second step of a CGGA (Cascade Golden-Gate and Gateway/Gibson Assemble method) according to an embodiment of the present invention
  • CGGA Cluster Golden-Gate and Gateway/Gibson Assemble method
  • Figure 15 is a diagram showing the effect of detecting a virus at a cellular level according to an embodiment of the present invention.
  • Figure 16 is a graph showing in vitro detection of cytokine activity (plasmid vector expressing cytokine) according to an embodiment of the present invention.
  • Figure 17 is a graph showing in vitro detection of cytokine activity (viral vector expressing cytokine) according to an embodiment of the present invention.
  • Figure 18 is a nude mouse model for treating HepG2, Huh7 and Hepa1-6 using oncolytic adenovirus according to an embodiment of the present invention
  • 19 is a C57 mouse model for treating Hepa1-6 using an oncolytic adenovirus according to an embodiment of the present invention.
  • Figure 20 is a graph showing the expression of T cell gamma interferon and Ki67 markers for oncolytic virus treatment for tumor infiltration according to an embodiment of the present invention
  • Figure 21 is a challenge experiment of mouse liver cancer cell Hepa1-6 for re-inoculation of mice after treatment according to an embodiment of the present invention
  • Figure 22 is a tumor-virus-immune system model in accordance with an embodiment of the present invention.
  • Figure 23 is a graph showing the rate of virus replication in a tumor-virus system simulating a minimum tumor volume under different initial tumor titers and initial tumor sizes under different initial tumor sizes, initial viral titers, and viral replication rates, in accordance with an embodiment of the present invention.
  • the tumor-virus system changes with time to the final state, the minimum value of the tumor volume changes;
  • Figure 24 is a graph showing the final state tumor volume under the influence of the tumor-virus-immunological system simulation immune cell on the different inhibitory effects of tumor and virus according to an embodiment of the present invention.
  • the invention proposes an expression system.
  • the expression system comprises: a first nucleic acid molecule comprising a 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 encoding a transcriptional activator; a third nucleic acid molecule comprising a first recognition sequence of the transcriptional activator; a fourth nucleic acid molecule, the fourth nucleic acid molecule
  • the third nucleic acid molecule is operably linked, the fourth nucleic acid molecule comprising a first promoter and a first regulatory element; a fifth nucleic acid molecule, the fifth nucleic acid molecule being operably linked to a fourth nucleic acid molecule,
  • the fifth nucleic acid molecule encodes a first regulatory protein; a sixth nucleic acid molecule comprising a second recognition sequence of the transcriptional activator; a seventh nucleic acid molecule
  • the cell-specific promoter is a tumor cell-specific promoter selected from the group consisting of an alpha-fetoprotein-specific promoter (AFP) and a Survivin gene promoter (SUR).
  • Human telomerase reverse transcriptase (hTERT) gene promoter cholecystokinin A receptor gene promoter (CCKAR promoter), carcinoembryonic antigen promoter (CEA promoter), proto-oncogene human epidermal growth factor receptor 2 Human epidermal growth factor receptor-2 (HER2), prostaglandin oxidase reductase 2 promoter (Cyclooxygenase 2, COX2), chemokine receptor-4 (CXCR4), E2F-1 gene Promoter (E2F-1promoter), mucin promoter (Mucin1MUC1), prostate specific antigen (Prostate-specific antigen PSA), human tyrosine related protein 1 (TRP1), tyrosinase promoter (ty)
  • the transcriptional activator is at least one selected from the group consisting of Gal4VP16, Gal4esn, Gal4VP64, dCas9-VP16, dCas9-VP64, dCas9-VPR, dCas9-VTR, and rtTA.
  • Yeast Gal4-UAS base Since the expression system is one of the most well understood eukaryotic transcriptional regulatory systems, the Gal4 gene encodes a transcriptional activator in yeast (Saccharomyces cerevisiae).
  • Gal4 recognizes a 17 bp long sequence of the upstream activation sequence (UAS) of the gene promoter: 5'-CGGRNNRCYNYNYNCNCCG-3' (R stands for ⁇ , Y stands for pyrimidine, and N stands for any deoxynucleotide). Gal4 acts with Gal80, which binds to galactose metabolites. Gal4 has two domains: a DNA binding domain (N-terminal domain) and an activation domain (C-terminal domain). It has a zinc cluster domain, specifically a Zn(2)-Cys(6) dual core cluster domain. The form in which it functions is often a homodimer.
  • UAS upstream activation sequence
  • Gal4 acts with Gal80, which binds to galactose metabolites.
  • Gal4 has two domains: a DNA binding domain (N-terminal domain) and an activation domain (C-terminal domain). It has a zinc cluster domain, specifically a Zn(2)-Cys(6) dual core cluster domain.
  • the core sequence that binds to the GAL4 protein in the UAS sequence is a four-copy tandem repeat, each copy containing 17 base pairs, and the sequence 5 '-CGGAGTACTGTCGTGGG-3'.
  • Gal4 is present in mammalian cells and activates the target gene only when UAS is recognized, and initiates downstream gene transcription with good specificity and inducibility.
  • the protein activation domain of Gal4 can be replaced by the activation domain of herpes simplex virus VP16 protein or 4 copies of VP16, VP64.
  • the fusion transcription factor thus formed increases the transcriptional activation activity of Gal4 protein while maintaining the binding specificity of Gal4 protein.
  • the tetracycline-inducing system is a bacterial-derived transcriptional activation system for eukaryotic cells.
  • the tetracycline response element contains 7 repeats of the 19-nucleotide tetracycline operon sequence 5'-TCCCTATCAGTGATAGAGA-3', which can be recognized by tetracycline-inducible proteins.
  • the tetracycline-inducing system includes a tetracycline-inducing inhibition system and a tetracycline-inducing activation system.
  • the tetracycline-inducing inhibition system comprises a tetracycline-responsive element and a tetracycline repressor tetR.
  • the tetracycline-inducible activation system comprises a tetracycline responsive element and a reverse tetracycline-controlled trans a ctivator rtTA.
  • rtTA When tetracycline is absent, rtTA cannot bind to the TRE element and cannot activate downstream gene expression; when tetracycline is present, rtTA binds to TRE in tetracycline to activate downstream gene expression.
  • dCas9 nuclease deactivated Cas9
  • dCas9 is a mutant form of Cas9 that recognizes the target sequence under gRNA-mediated, but dCas9 has no cleavage activity on the target sequence, and only specifically recognizes the activity of binding DNA.
  • dCas9 can be expressed in fusion with various activation elements, it can effectively activate the expression of the target gene.
  • the first recognition sequence and the second recognition sequence are each independently selected from at least one of a target sequence of 5 ⁇ UAS, 7 ⁇ tetO and dCas9.
  • the 5 ⁇ UAS is the response element of the 5 copies of the Gal4VP16 regulatory system.
  • the core sequence that binds to the Gal4 protein in the UAS sequence is a four-copy tandem repeat, each copy containing 17 base pairs and the sequence is 5 '-CGGAGTACTGTCGTGGG-3'.
  • 7 ⁇ tetO is a 7-copy TRE that is the recognition sequence for the response element of the tetracycline regulatory system.
  • the tetracycline response element contains 7 repeats of the 19-nucleotide tetracycline operon sequence 5'-TCCCTATCAGTGATAGAGA-3', which can be recognized by tetracycline-inducible proteins.
  • the first promoter and the second promoter are miniCMV.
  • 5 ⁇ UAS-miniCMV is a synthetic inducible promoter.
  • 5 ⁇ UAS is the recognition sequence of Gal4 protein
  • miniCMV is a partial sequence of the promoter of Cytomegalovirus.
  • TRE is also an inducible promoter.
  • 7 ⁇ tetO-miniCMV, tetO is the recognition sequence of the response element of the tetracycline system.
  • the first regulatory protein and the second regulatory protein are each independently selected from at least one of Lacl, tetR, zinc finger, KRAB, dCas9-KRAB.
  • Lactose repressor LacI
  • Lactose operon Lactoseoperon LacO
  • the base is closely linked to the base sequence of the LacO minor groove. This tight binding prevents the high affinity binding of RNA polymerase from entering the extended state in the promoter region, thereby preventing mRNA transcription and expression of downstream genes.
  • the tetracycline repressor tetR TetR contains a conserved helix-turn-helix DNA binding region (forming a homodimer) that binds to the tetracycline operon tetO and inhibits the transcriptional expression of downstream genes.
  • Zinc finger zinc finger protein consists of a ring containing about 30 amino acids and a Zn 2+ coordinated to 4 Cys or 2 Cys and 2 His on the ring, forming a structure like a finger.
  • Zinc finger proteins are a class that play an important role in gene regulation. According to their conserved domains, zinc finger proteins can be mainly classified into C2H2, C4 and C6.
  • Zinc refers to the regulation of gene expression, cell differentiation and embryonic development at the transcriptional and translational levels by specific binding to the target molecule DNA, RNA, DNA-RNA, and to its own or other zinc finger proteins.
  • the Krüppel associated box (KRAB) domain is a class of transcriptional repressor regions present in nearly 400 zinc finger proteins in humans. KRAB generally consists of 75 amino acid residues and the smallest inhibitory effective region consists of 45 amino acids. It functions by means of an amphipathic helical connection between proteins. It can be divided into two regions, A and B, which are encoded by different exons, wherein A box plays a central role in transcriptional repression, and B box enhances the transcriptional repression function of A box.
  • dCas9 nuclease deactivated Cas9
  • dCas9 is a mutant form of Cas9 that recognizes the target sequence under gRNA-mediated, but dCas9 has no cleavage activity on the target sequence, and only specifically recognizes the activity of binding DNA.
  • dCas9 and the suppressor region are expressed, the expression of the target gene can be effectively inhibited.
  • the first regulatory element and the second regulatory element are each independently selected from at least one of a tetO, a LacO, a zinc finger target site, and a target sequence of dCas9.
  • the tetO (tetracycline operon tetO) tetracycline operon can be recognized by tetracycline inhibitors to inhibit the expression of downstream genes.
  • the LacO (Lactose operon LacO) lactose operon can be recognized by the lactose repressor (Lactose repressor LacI) to inhibit the expression of downstream genes.
  • the zinc finger target site can be recognized by zinc finger proteins to regulate the expression of downstream genes.
  • the dCas9 target sequence can be recognized by dCas9-KRAB with the aid of a complementary gRNA and inhibits the expression of downstream genes.
  • the first regulatory protein is LacI
  • the second regulatory element comprises a plurality of repeating LacO sequences, at least one of the plurality of repeated LacO sequences being set at the second promoter Downstream.
  • LacI table Upon arrival, the LacO sequence can be specifically bound, thereby inhibiting the function of the second promoter.
  • the Lactose repressor LacI recognizes the specific sequence by the helix-turn-helical element of the DNA binding domain and the large groove of the lactose operon (Lactoseoperon LacO). Binding, while the symmetry-dependent ⁇ -helix residue is closely linked to the base sequence of the LacO minor groove.
  • the inventors constructed LacO on both sides of the miniCMV sequence of the 5 ⁇ UAS-miniCMV promoter, and it was experimentally shown that the system can effectively inhibit the expression of genes downstream of the promoter. However, the inventors do not preclude the use of other suppression systems at this location.
  • the second regulatory protein is tetR-KRAB
  • the first regulatory element comprises a plurality of repeated tetO sequences, at least one of the plurality of repeated tetO sequences being set at the first Downstream of the promoter.
  • the inhibition system selected in the embodiment of the present invention is the tetR-KRAB/tetO system
  • the tetracycline repressor tetR TetR contains a conserved helix-turn-helix DNA binding region (forming a homodimer), which binds to the tetracycline operon. (tetracycline operon tetO) inhibits transcriptional expression of downstream genes.
  • the inventors constructed tetO on both sides of the miniCMV sequence of the 5 ⁇ UAS-miniCMV promoter, and it was shown by experiments that the system can effectively inhibit the expression of genes downstream of the promoter.
  • the switching system composed of the first regulatory protein and the second regulatory protein can effectively input signals correspondingly and effectively distinguish different cell lines.
  • the inventors here do not preclude the use of other suppression systems.
  • At least one of the fifth nucleic acid molecule and the ninth nucleic acid molecule further comprises a sequence encoding a protein of interest.
  • the fifth nucleic acid molecule comprises a sequence encoding the protein of interest, and the protein of interest comprises at least one selected from the group consisting of a viral replication packaging protein and an immune effector.
  • the viral replication packaging protein can effectively ensure the survival and replication of the expression system vector in the host; the expression of the immune effector can effectively activate the body's immune system, thereby promoting immune killing of specific cells such as tumor cells.
  • the virus replication packaging-related protein comprises at least one selected from the group consisting of an adenovirus E1 gene, an E1A gene, an E1B gene, an E2 gene, and an E4 gene.
  • adenoviral genes are mainly divided into early expression genes (E1 ⁇ 4) and late expression genes (L1 ⁇ 5). After the adenoviral genome enters the nucleus, the cellular transcription factor first binds to the enhancer upstream of the E1A region, expressing the E1A protein, which regulates cellular metabolism and makes the viral DNA more easily replicated in the cell.
  • the E1A protein also activates promoters of other early genes (E1B, E2A, E2B, E3, and E4), where E2B drives three additional early gene transcriptional terminal protein precursors (pTPs) involved in viral replication.
  • E1B Single-stranded DNA binding proteins
  • DNA pol DNA polymerase
  • the E1 region gene expression products are divided into E1A and E1B.
  • E1A is mainly composed of two components, 289R (or 13S) and 243R (or 12S).
  • E1A protein The main function of the E1A protein is to regulate cellular metabolism, making cells more susceptible to viral replication.
  • E1B19K is homologous to the expression product of the Bcl-2 gene, and can prevent apoptosis or necrosis of cells by inactivating and clearing Bax family members.
  • the E1B55K gene product can down-regulate the transcription level of the p53 gene, and other adenoviral genes (such as E4 or f6) are involved in this process.
  • the E1B55K gene product is also involved in viral replication, transcription of viral late mRNA, and viral RNA transport.
  • the E2 region gene expression products are divided into E2A and E2B.
  • E2A is DNA Binding Protein (DBP); two main products of E2B are terminal protein precursor (pTP) and viral DNA polymerase (pol).
  • the three proteins interact with at least three intracellular factors to initiate adenoviral DNA replication and transcription and translation of the viral late genes.
  • the gene product of the E4 region is commonly referred to as orf 1 to 6/7 and is mainly involved in the metabolism of viral messenger RNA. There is also the function of promoting viral DNA replication and shutting down host protein synthesis. Studies have found that some E4 products can bind to DNA-activated protein kinases to prevent viral DNA from concatenating. Since this kinase activates the p53 gene, it is believed that some E4 region gene products can inhibit apoptosis. Many E1B and E4 gene products are involved in antagonizing E1A protein function. For example, E4 inhibits E1A activation of the E2F promoter.
  • the immune effector comprises a sequence selected from the group consisting of 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, and GM.
  • - at least one of CSF, IL-2, IL-12, IL-15 or a fusion expression form of these factors.
  • PD-1 (programmed death 1) programmed death receptor 1 is an important immunosuppressive molecule.
  • PD-1 is an important inhibitory molecule on the surface of T cells, and its intracellular domain contains an immunoreceptor tyrosine inhibitory motif ITIM and an immunoreceptor tyrosine transfer motif (ITSM).
  • ITSM mediates the recruitment of protein tyrosine phosphatase family phosphatase and the inhibition of T cell activation signals.
  • Its ligands are PD-L1 and PD-L2, which play a major role in suppressing T cells in the tumor microenvironment of the immune system. The important role of activation.
  • Programmed cell death 1 ligand also known as cluster of differentiation 274 (CD274) or B7 homolog (B7homolog 1, B7-H1), is human A protein in the body that is encoded by the CD274 gene.
  • PD-L1 is inducible in hematopoietic cells in a variety of tumor cells and tumor cell microenvironments. Its expression level is positively correlated with the malignancy of some tumors.
  • PD-1 antibody and PD-L1 antibody can block the binding of PD-1 to PD-L1, and tissue tumor cells escape from the PD-1/PD-L1 pathway.
  • CTLA-4 Cytotoxic T lymphocyte associate protein-4, which is a type of co-stimulatory molecule expressed on the surface of T cells. Similar to the function of CD28, CTLA-4 is capable of specifically binding to CD80/CD86 on the surface of APC to activate downstream signals during T cell activation.
  • Treg regulatory T cells
  • Activation is accompanied by severe autoimmune diseases.
  • CTLA-4 is also expressed in conT cells, and its role is to inhibit the signaling of T cell activation.
  • T cell immunoglobulin and mucin-domain containing molecule (TIM) gene family was discovered by Mclntire in 2001 when searching for mouse asthma susceptibility genes and was identified by genomic analysis and cloning. A new family of genes identified that contain immunoglobulin V regions and mucin regions. Tim-3 is an important member of the TIM family and expresses a negative regulatory molecule on the surface of activated Th1 cells. Expression of TIM3 has also been found in CD8 + T cells, Th17 cells, Treg, NK cells, and other lymphocyte subpopulations.
  • GM-CSF granulocyte-macrophage Colony Stimulating Factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • T cells T cells
  • NK cells NK cells
  • GM-CSF stimulates stem cells to produce granules.
  • Cells neutralils, eosinophils, and basophils and monocytes.
  • GM-CSF also affects mature cells in the immune system, such as inhibiting the transfer of neutrophils and altering receptor expression on the cell surface. This factor also inhibits fungal infections by activating macrophages.
  • IL-2 Interleukin 2
  • Interleukin 2 mediates cytokines between white blood cells and white blood cells and between white blood cells and other cells.
  • IL-2 is mainly produced by activated T cells, and acts on local target cells in an autocrine and paracrine manner. It is a major cytokine involved in the immune response and has obvious immune effects. It can promote T cell proliferation and produce cytokines, promote B cell proliferation and secretion of Ig, activate macrophages, and enhance NK cell activation and proliferation.
  • IL-2 also has a negative regulation effect, which can induce apoptosis of Ag-activated T cells, limit the intensity of immune response, and avoid obvious immune damage.
  • Interleukin-12 is a kind of leukocyte-mediated by dendritic cells, macrophages, neutrophils, and human B lymphoblasts (nc-37) in response to antigen stimulation.
  • Prime. IL-12 called T cell stimulating factor, participates in the differentiation of naive cells into Th1 and stimulates the production of interferon gamma (IFN- ⁇ ) and tumor necrosis factor alpha (TNF- ⁇ ).
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-12 plays an important role in regulating the activity of natural killer cells and T lymphocytes.
  • IL-12 mediates enhanced cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes.
  • IL-12 also has anti-angiogenic activity, which means it blocks the formation of new blood vessels.
  • IP-10 protein 10
  • CXCL10 protein 10
  • IL-12 has been tested as a possible anti-cancer drug.
  • IL-15 is a recently discovered factor that can be produced by a variety of cells such as activated monocytes-macrophages, epidermal cells, and fibroblasts. The molecular structure of IL-15 is much similar to that of IL-2, so that the ⁇ -chain and the ⁇ -chain of the IL-2 receptor can be utilized to bind to target cells to exert a biological activity similar to IL-2.
  • IL-15 can induce B cell proliferation and differentiation, and is the only cytokine that can partially replace IL-2 to induce initial antibody production; IL-15 can stimulate T cell and NK cell proliferation, induce LAK cell activity, and can also interact with IL-12. Synergistically stimulate NK cells to produce IFN- ⁇ .
  • the target protein and the first regulatory protein are co-expressed under the control of the same promoter, and the cleavable linker peptide is passed between the target protein and the first regulatory protein. connected.
  • the target protein and the first regulatory protein are regulated and expressed under the same promoter, and the expression is cleaved at the linker peptide, and the target protein is The first regulatory protein is separated, and the target protein and the first regulatory protein function independently of each other.
  • the ninth nucleic acid molecule and the tenth nucleic acid molecule independently inhibit expression of the first regulatory protein or the second regulatory protein, respectively, by the ninth and tenth nucleic acid molecules
  • the expression of the first and second regulatory proteins is inhibited by RNA interference, wherein the microRNA is a specific microRNA expressed by different cellular microenvironments, and the ninth or tenth nucleic acid molecule is a specific target sequence of the microRNA, thereby further interfering with RNA interference
  • the specific effect of the microRNA expressed in the specific microenvironment on the target sequence can be achieved, thereby achieving specific regulation of the expression of the first regulatory protein or the second regulatory protein.
  • MicroRNAs are a class of endogenous small RNAs of about 20-24 nucleotides in length, and several miRNAs can regulate the same gene. The expression of a gene can be finely regulated by a combination of several miRNAs. MicroRNA exists in many forms, the most primitive is pri-miRNA, which is about 300-1000 bases in length; after a single processing, pri-miRNA becomes a pre-miRNA, a microRNA precursor, with a length of about 70-90 bases. The pre-miRNA is digested with Dicer to form a mature miRNA of about 20 to 24 nt in length. The RNA-induced silencing complex (RISC) inhibits the expression of target genes.
  • RISC RNA-induced silencing complex
  • microRNA-RISC effect of microRNA-RISC on target gene mRNA has always been largely dependent on its degree of complementation with the target gene transcript sequence, in three ways. The first is to cleave the mRNA molecule of the target gene - the miRNA is fully complementary to the target gene, and its mode of action and function are very similar to those of the siRNA, and finally the target mRNA is cleaved. In plants, most of the miRNAs in this way, after the target gene mRNA is broken, the poly(A)-free molecule has multiple U's at the 3' end and is rapidly degraded, and the poly(A)-containing molecule can be stably present. For a while (eg Arabidopsis miR-171).
  • miRNAs There is currently one miRNA in the plant that is fully complementary to the three potential target genes, although it is unclear whether these genes are targets of miRNAs. This is the first time that miRNAs are fully complementary to their potential targets, suggesting that miRNAs May contain a similar mode of action as siRNA. The second is to inhibit the translation of the target gene, which does not completely complement the target gene, thereby suppressing translation without affecting the stability of the mRNA. This miRNA is the most widely discovered species (such as nematode lin-4). A very small number of miRNAs in plants inhibit the target gene in this way. The third is binding inhibition - having the above two modes of action: direct binding to cleavage of mRNA when complementary to the target gene; and regulation of gene expression when not intimately bound to the target gene.
  • the ninth nucleic acid molecule comprises a nucleic acid sequence specifically recognized by a first microRNA, the tenth nucleic acid molecule comprising a nucleic acid sequence specifically recognizing a second microRNA, the first microRNA being a normal cell Specific microRNAs, the second microRNA is an abnormal cell-specific microRNA.
  • the first regulatory protein is expressed in abnormal cells, and is not expressed or underexpressed in normal cells, while the second regulatory protein is expressed in normal cells and is not expressed or expressed in abnormal cells.
  • the first microRNA comprises at least one selected from the group consisting of miR199a, miR95, miR125, miRamir25b, Let-7, miR143, miR145, and miR200C.
  • the above microRNA is expressed in normal liver cells.
  • the second microRNA comprises at least one selected from the group consisting of miR21, miR223, miR224, miR221, miR18, miR214, miR146a, and miR1792 expressed in hepatoma cells (HepG2, Huh7, PLC) .
  • the second microRNA is a microRNA specifically expressed by a liver cancer cell.
  • the first regulatory protein is expressed in liver cancer cells, and is not expressed or expressed in normal cells, while the second regulatory protein is expressed in normal cells and is not expressed or expressed in liver cancer cells.
  • the first nucleic acid molecule and the second nucleic acid molecule are supported on a first expression vector
  • the selected ninth nucleic acid molecule is supported on a second expression vector
  • the sixth nucleic acid molecule, the seventh nucleic acid molecule, the eighth nucleic acid molecule, and optionally the tenth nucleic acid molecule are loaded in a third On the expression vector.
  • the first, second and third expression vectors serve as a loading vector for the expression system to effect regulation of the specific expression of the gene of interest in a suitable microenvironment, such as a cell.
  • the selection of the expression vector is not particularly limited as long as the function of the expression system in a suitable microenvironment can be achieved.
  • the first expression vector, the second expression vector and the third expression vector are each independently selected from at least one of the following:
  • the liposome carrier is a liposome-DNA complex formed by packaging DNA with an artificial bilayer phospholipid.
  • the liposome carrier is non-toxic and non-antigenic, and the target gene and the liposome are encapsulated to be protected from nuclease degradation, and the capacity is large, and it can be used alone or in combination with other carriers, and the target gene can be introduced into a specific site by intravenous injection. Disadvantages: short expression time, can not pass the cell membrane barrier.
  • Molecularly coupled carrier The molecularly coupled carrier consists of three parts: DNA, DNA binding factor and ligand.
  • the virus comprises at least one selected from the group consisting of an adenovirus, a vaccinia virus, and a retrovirus.
  • the first expression vector, the second expression vector and the third expression vector are the same vector.
  • the inventors load the components in the expression system of the present application on the same expression vector by Cascade Golden-Gate Gibson/Gateway Assemble Method technology, and the plurality of large fragments can be effectively solved by being loaded on the same expression vector.
  • the carrier has a very low transfection efficiency.
  • the same vector is an adenovirus.
  • Adenovirus as a gene therapy vector has the following advantages: 1) a wide range of hosts and low pathogenicity to humans.
  • Adenoviral vector systems are widely used for the expression of human and non-human proteins.
  • Adenoviruses can infect a range of mammalian cells and are therefore useful for expression of recombinant proteins in most mammalian cells and tissues. It is particularly important to note that adenoviruses are epithelial, whereas most human tumors are derived from epithelial cells.
  • the replication genes and pathogenic genes of adenoviruses are quite clear and have been prevalent in the human population (70-80% of adults have neutralizing antibodies to adenovirus).
  • Retroviruses can only infect proliferating cells, so DNA transfection cannot be performed in non-proliferating cells, but cells must be maintained in a continuous culture.
  • Adenovirus can infect almost all cell types, except for some anti-adenovirus-infected lymphoma cells.
  • Adenovirus is the best system for studying gene expression in primary non-proliferating cells, which allows direct comparison between the results obtained in transformed cells and primary cells.
  • It can effectively proliferate and has a high titer.
  • the adenovirus system produces 10 10 to 10 11 VP/mlL and is concentrated up to 10 13 VP/mL, making it ideal for gene therapy.
  • 4) is homologous to human genes.
  • Adenoviral vector systems generally use human viruses as vectors and human cells as hosts, thus providing an ideal environment for accurate post-translational processing and proper folding of human proteins. Most human proteins achieve high levels of expression and are fully functional.
  • 5) Not integrated into the chromosome, no insertional mutagenicity. Retroviruses can be randomly integrated into the host chromosome, resulting in gene inactivation or activation of oncogenes. The adenovirus does not integrate into the chromosome in almost all known cells except the egg cell, and thus does not interfere with other host genes.
  • Integrating a single copy of the virus in an egg cell is a better system for producing transgenic animals with specific characteristics.
  • the inventors injected the above adenovirus into a tumor mouse model to significantly inhibit the growth of mouse tumors.
  • the above adenovirus can be used as a safe and effective oncolytic virus vaccine to achieve safe and effective specific killing of related tumors.
  • the adenovirus is obtained by removing the E1 gene and a part of the E3 gene associated with adenoviral replication packaging, and the E1A gene is constructed by a stepwise Golden Gate method.
  • the gene line is finally inserted into the adenoviral vector by way of Gateway or Gibson.
  • the inventors inserted a gene line recognizing cancer cells into an adenovirus vector in three steps.
  • a marker element that distinguishes normal cells from cancer cells such as a tumor-specific promoter, a suppressor element related to gene line construction, and a recognition sequence of a microRNA that distinguishes cancer cells, is constructed on a primary vector plasmid to constitute a primary component library.
  • Esp3I recognition sites are designed at both ends of the component for constructing multiple primary components onto the secondary expression system by means of Golden Gate.
  • the expression system library is constructed, and multiple first-level components are selected from the first-level library to assemble them into three expression systems by means of Golden Gate, including a tumor-specific promoter system, which is mediated by the first regulatory element.
  • the inhibition system mediated by the inhibition system and the second regulatory element.
  • the recognition site of BsaI endonuclease was designed at both ends of each expression system.
  • the gene lines are assembled, and the three expression systems are randomly assembled by the Golden Gate method to form a complete gene line. Both ends of the gene line were designed with Gibson homologous sequences or Gateway recognition sites for further experiments.
  • the gene line was constructed by Gibson or Gateway to an adenoviral vector in which E1 and part E3 have been removed.
  • the above-described manner of obtaining an adenovirus enables rapid modification of a complex, large-fragment oncolytic adenoviral vector.
  • the invention proposes a recombinant virus.
  • the recombinant virus comprises: a first nucleic acid molecule, the first nucleic acid molecule comprising a tumor cell-specific promoter, and the tumor cell-specific promoter is an alpha-fetoprotein-specific promoter; a nucleic acid molecule operably linked to the first nucleic acid molecule, the second nucleic acid molecule encoding a transcriptional activator, the transcriptional activator is Gal4VP16; a third nucleic acid molecule, the third The nucleic acid molecule comprises a first recognition sequence of the transcriptional activator, the first recognition sequence being 5 x UAS; a fourth nucleic acid molecule operably linked to the third nucleic acid molecule, The fourth nucleic acid molecule comprises a first promoter and a first regulatory element, the first promoter is a miniCMV, and the first regulatory element comprises a plurality of repeating tet
  • the first regulatory protein LacI and the target protein are specifically expressed in tumor cells
  • the second regulatory protein tetR-KRAB is specifically not expressed or underexpressed in tumor cells, and thus the inhibition mechanism of the first promoter miniCMV mediated by tetR-KRAB is released, the first regulatory protein LacI, the target protein in the first promoter miniCMV start up Under the regulation of effective expression, the LacI-mediated inhibition mechanism effectively inhibited the function of the second promoter miniCMV, and the expression of tetR-KRAB was further inhibited.
  • the recombinant virus according to an embodiment of the present invention, more specific expression of the protein in the tumor cell (such as the target protein E1A, the first regulatory protein LacI) or no expression (such as the second regulatory protein tetR-KRAB) can be achieved.
  • the expression or non-expression efficiency and specificity in tumor cells are higher.
  • the recombinant virus is at least one selected from the group consisting of a retrovirus, an adenovirus, a herpes virus, and a vaccinia virus.
  • a retrovirus an adenovirus
  • a herpes virus a vaccinia virus.
  • vaccinia virus a retroviral vector
  • Characteristics of adenovirus vector wide range of host; adenoviral protein expression is not necessary for host proliferation; high virus titer can be obtained; recombinant is very stable; no tumor is caused; high safety; no capsule It is not easily inactivated by complement and can be directly applied in the body; it is not integrated into the chromosome.
  • Characteristics of herpes simplex virus vector high titer; large capacity; both proliferating cells and non-proliferating cells can be infected; unintegrated, but long-term existence and stable expression.
  • the recombinant virus is an adenovirus.
  • adenovirus has many advantages as a gene therapy vector: 1. The host has a wide range and is low in pathogenicity to humans. Adenoviral vector systems are widely used for the expression of human and non-human proteins. Adenoviruses can infect a range of mammalian cells and are therefore useful for expression of recombinant proteins in most mammalian cells and tissues. It is particularly important to note that adenoviruses are epithelial, whereas most human tumors are derived from epithelial cells.
  • adenovirus In addition, the replication genes and pathogenic genes of adenoviruses are quite clear and have been prevalent in the human population (70-80% of adults have neutralizing antibodies to adenovirus). Human infection with wild-type adenovirus produces only mild self-limiting symptoms, and ribavirin is effective. 2. Infect and express genes in proliferating and non-proliferating cells. Retroviruses can only infect proliferating cells, so DNA transfection cannot be performed in non-proliferating cells, but cells must be maintained in a continuous culture. Adenovirus can infect almost all cell types, except for some anti-adenovirus-infected lymphoma cells.
  • Adenovirus is the best system for studying gene expression in primary non-proliferating cells, which allows direct comparison between the results obtained in transformed cells and primary cells. 3. It can effectively proliferate and has a high titer. The adenovirus system produces 10 10 to 10 11 VP/mL and is concentrated up to 10 13 VP/mlL, making it ideal for gene therapy. 4. Is homologous to human genes. Adenoviral vector systems generally use human viruses as vectors and human cells as hosts, thus providing an ideal environment for accurate post-translational processing and proper folding of human proteins. Most human proteins achieve high levels of expression and are fully functional. 5. Not integrated into the chromosome, no insertional mutagenicity.
  • Retroviruses can be randomly integrated into the host chromosome, resulting in gene inactivation or activation of oncogenes.
  • the adenovirus does not integrate into the chromosome in almost all known cells except the egg cell, and thus does not interfere with other host genes. Integrating a single copy of the virus in an egg cell is a better system for producing transgenic animals with specific characteristics.
  • 6. Can be expanded in suspension culture. 293 cells can be adapted to suspension culture, and this adjustment allows the virus to be expanded in large quantities. A large number of facts have shown that suspension 293 cells can express recombinant proteins in a 1-20 L bioreactor. 7. Can express multiple genes at the same time. This is the first expression system that can be used to design multiple genes in the same cell line or tissue.
  • the simplest method is to insert a double expression cassette containing two genes into an adenovirus transfer vector, or co-transfect a target cell strain with a different recombinant virus to express a protein separately. Determination of the MOI ratio of different recombinant viruses can correctly estimate the relative co-expression of each recombinant protein.
  • the immune effector comprises a restriction sequence selected from the group consisting of an antagonistic PD-1 gene, an inhibitory sequence which antagonizes the PD-L1 gene, an inhibitory sequence which antagonizes the CTLA4 gene, an inhibitory sequence which antagonizes the Tim-3 gene, and IL. - 2, at least one of IL-12, IL-15, GM-CSF or a fusion expression form of these factors.
  • PD-1 programmeed death 1 is a major immunosuppressive molecule.
  • PD-1 is an important inhibitory molecule on the surface of T cells, and its intracellular domain contains an immunoreceptor tyrosine inhibitory motif ITIM and an immunoreceptor tyrosine transfer motif (ITSM).
  • ITSM mediates the recruitment of protein tyrosine phosphatase family phosphatase and the inhibition of T cell activation signals.
  • Its ligands are PD-L1 and PD-L2, which play a major role in suppressing T cells in the tumor microenvironment of the immune system. The important role of activation.
  • Programmed cell death 1 ligand also known as cluster of differentiation 274 (CD274) or B7 homolog (B7homolog 1, B7-H1), is human A protein in the body that is encoded by the CD274 gene.
  • PD-L1 is inducible in hematopoietic cells in a variety of tumor cells and tumor cell microenvironments. Its expression level is positively correlated with the malignancy of some tumors.
  • PD-1 antibody and PD-l1 antibody can block the binding of PD-1 to PD-L1, and tissue tumor cells escape from the PD-1/PD-L1 pathway.
  • CTLA-4 Cytotoxic T lymphocyte associate protein-4, which is a type of co-stimulatory molecule expressed on the surface of T cells. Similar to the function of CD28, CTLA-4 is capable of specifically binding to CD80/CD86 on the surface of APC to activate downstream signals during T cell activation.
  • Treg regulatory T cells
  • Activation is accompanied by severe autoimmune diseases.
  • CTLA-4 is also expressed in conT cells, and its role is to inhibit the signaling of T cell activation.
  • T cell immunoglobulin and mucin-domain containing molecule (TIM) gene family was discovered by Mclntire in 2001 when searching for mouse asthma susceptibility genes and was identified by genomic analysis and cloning. A new family of genes identified that contain immunoglobulin V regions and mucin regions.
  • TIM3 is an important member of the TIM family and expresses a negative regulatory molecule on the surface of activated Th1 cells. Expression of TIM3 has also been found in CD8 + T cells, Th17 cells, Treg, NK cells, and other lymphocyte subpopulations.
  • GM-CSF granulocyte-macrophage Colony Stimulating Factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • T cells T cells
  • NK cells NK cells
  • GM-CSF stimulates stem cells to produce granules.
  • Cells neutralils, eosinophils, and basophils and monocytes.
  • GM-CSF also affects mature cells in the immune system, such as inhibiting the transfer of neutrophils and altering receptor expression on the cell surface. This factor also inhibits fungal infections by activating macrophages.
  • IL-2 Interleukin 2
  • Interleukin 2 mediates cytokines between white blood cells and white blood cells and between white blood cells and other cells.
  • IL-2 is mainly produced by activated T cells, and acts on local target cells in an autocrine and paracrine manner. It is a major cytokine involved in the immune response and has obvious immune effects. It can promote T cell proliferation and produce cytokines, promote B cell proliferation and secretion of Ig, activate macrophages, and enhance NK cell activation and proliferation.
  • IL-2 also has a negative regulation effect, which can induce apoptosis of Ag-activated T cells, limit the intensity of immune response, and avoid obvious immune damage.
  • Interleukin-12 is a kind of leukocyte-mediated by dendritic cells, macrophages, neutrophils, and human B lymphoblasts (nc-37) in response to antigen stimulation.
  • Prime. IL-12 called T cell stimulating factor, participates in the differentiation of naive cells into Th1 and stimulates the production of interferon gamma (IFN- ⁇ ) and tumor necrosis factor alpha (TNF- ⁇ ).
  • IFN- ⁇ interferon gamma
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-12 plays an important role in regulating the activity of natural killer cells and T lymphocytes.
  • IL-12 mediates enhanced cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes.
  • IL-12 also has anti-angiogenic activity, which means it blocks the formation of new blood vessels.
  • IP-10 protein 10
  • CXCL10 protein 10
  • IL-12 has been tested as a possible anti-cancer drug.
  • IL-15 is a recently discovered factor that can be produced by a variety of cells such as activated monocytes-macrophages, epidermal cells, and fibroblasts. The molecular structure of IL-15 is much similar to that of IL-2, so that the ⁇ -chain and the ⁇ -chain of the IL-2 receptor can be utilized to bind to target cells to exert a biological activity similar to IL-2.
  • IL-15 can induce B cell proliferation and differentiation, and is the only cytokine that can partially replace IL-2 to induce initial antibody production; IL-15 can stimulate T cell and NK cell proliferation, induce LAK cell activity, and can also interact with IL-12. Synergistically stimulate NK cells to produce IFN- ⁇ .
  • the invention proposes a recombinant cell.
  • the recombinant cell comprises an expression system as described above.
  • the recombinant cell according to the embodiment of the invention can effectively activate the systemic immune response of the human body, and attack the xenogeneic cells, such as tumor cells, with high safety and specificity.
  • At least a portion of the expression system is integrated into the genome of the recombinant cell.
  • the expression system replicates as the recombinant cell genome replicates, and the expression system continues to be effective in regulating the expression of the protein of interest.
  • the present invention provides the use of the aforementioned expression system, the recombinant virus described above, and the aforementioned recombinant cells for the preparation of a medicament for the treatment of cancer.
  • the expression system described in the present application can achieve specific expression of a target protein in tumor cells, and the drug according to the embodiment of the present application is more effective, specific, and safer for treating cancer.
  • the inventors used the concept of synthetic biology to design a gene line that responds to multiple targets to regulate the expression of the key gene E1A associated with adenoviral transcription.
  • a related cytokine or antibody gene that activates a systemic immune response is co-expressed.
  • the gene lines designed by the inventors can regulate the packaging of adenoviruses at multiple levels.
  • the inventors used a tumor-specific promoter to regulate
  • the microRNA target sequence inside the gene line can distinguish tumor cells from non-tumor cells in response to the expression of microRNAs in different cell lines.
  • the inventor designed the gene line to adopt a double suppression system that is very stable and can efficiently respond to external input signals such as cell-specific promoters and microRNA signals, and the gene line can further expand the difference of input signals and be more efficient. Differentiate between tumor cells and non-tumor cells.
  • the system removes the E1B gene of the adenovirus, and the E1B gene can interact with the P53 gene of the normal cell to ensure the smooth proliferation of the adenovirus in the cell.
  • the adenovirus could not proliferate in normal cells expressing P53.
  • the expression of the P53 gene is deleted in many tumor cells, and the adenovirus that does not express E1B can still be efficiently expanded. Therefore, the adenoviral vector of the present embodiment is more specific and safer than the traditional oncolytic adenovirus by multi-angle and multi-level regulation.
  • this embodiment also utilizes this adenovirus as a vector to carry a variety of cytokines and antibody genes. At the same time as the oncolytic effect of adenovirus, these co-expressed factors are used to activate the systemic immune response, thereby further improving the effect of oncolytic adenovirus.
  • the present patent uses synthetic biology to construct a gene line to regulate the specific expression packaging of oncolytic adenovirus in hepatocellular carcinoma.
  • the adenovirus designed and constructed by the inventors is also loaded with cytokines that can activate the immune response of the body. By expressing these cytokines, the body can stimulate the immune response that inhibits tumor growth to achieve the purpose of treating tumors.
  • the system designed by this patent has great innovation and technical advantages:
  • the gene line designed in this patent comprehensively utilizes multi-level and multi-level biomarkers to distinguish normal cells from cancer cells, and uses double-inhibited closed-loop gene lines to regulate the replication of adenovirus in different cells. Through this genetic line, it is more sensitive to respond to different cell line microenvironments while reducing the effects of gene expression noise.
  • This patent utilizes the gene line to carry a variety of cytokines and/or antibody sequences to achieve an effective immune response in the cells of interest.
  • the biomarkers involved in the genetic lines are modularized, and each module is built into a library containing biomarkers for different diseases. Establish a genetic line for specific conditions by quickly building up relevant modules.
  • the cancer comprises liver cancer, lung cancer, colorectal cancer, melanoma, breast cancer or prostate cancer.
  • the inventors have found that the therapeutic effect of the drug according to the embodiment of the present invention on liver cancer, lung cancer, colorectal cancer, melanoma, breast cancer or prostate cancer is more remarkable.
  • microRNAs in different tumor cell lines is shown in Table 1.
  • the upward arrow indicates microRNAs that are highly expressed in cancer cells compared to normal cells
  • the downward arrow indicates microRNAs that are underexpressed in cancer cells compared to normal cells.
  • the invention proposes a method of expressing a protein of interest using an expression system, which is the expression system described above.
  • the method comprises: (1) causing the fifth nucleic acid molecule to comprise a nucleic acid sequence encoding the protein of interest; (2) inhibiting the tenth nucleic acid molecule from the second regulatory protein Expression to express the protein of interest.
  • the second regulatory protein-mediated inhibition mechanism of the first promoter is released, and the target protein is co-operated by the cell-specific promoter and the first promoter. Highly expressed in specific cells.
  • the expression is carried out in a cell.
  • the cells can provide a microenvironment for the expression of the protein of interest, and the expression of the protein of interest in the cell is more efficient.
  • the tenth nucleic acid molecule comprises a nucleic acid sequence specific for the second microRNA, and in step (2) comprises contacting the second microRNA with the tenth nucleic acid molecule.
  • MicroRNAs are a class of endogenous small RNAs of about 20-24 nucleotides in length. Multiple miRNAs can also regulate the same gene, and the expression of a single gene can be finely regulated by a combination of several miRNAs. MicroRNA exists in many forms, the most primitive is pri-miRNA, which is about 300-1000 bases in length; after a single processing, pri-miRNA becomes a pre-miRNA, a microRNA precursor, with a length of about 70-90 bases.
  • the pre-miRNA is digested with Dicer to form a mature miRNA of about 20 to 24 nt in length.
  • MicroRNA-mediated silencing complex RISC inhibits the expression of target genes.
  • the effect of microRNA-RISC on target gene mRNA has always been largely dependent on its degree of complementation with the target gene transcript sequence, in three ways. The first is to cleave the mRNA molecule of the target gene - the miRNA is fully complementary to the target gene, and its mode of action and function are very similar to those of the siRNA, and finally the target mRNA is cleaved.
  • the poly(A)-free molecule In plants, most of the miRNAs in this way, after the target gene mRNA is broken, the poly(A)-free molecule has multiple U's at the 3' end and is rapidly degraded, and the poly(A)-containing molecule can be stably present.
  • miRNAs may contain a similar mode of action as siRNA. The second is to inhibit the translation of the target gene, which does not completely complement the target gene, thereby suppressing translation without affecting the stability of the mRNA.
  • This miRNA is the most widely discovered species (such as nematode lin-4). A very small number of miRNAs in plants inhibit the target gene in this way.
  • the third is binding inhibition - with the above two modes of action: when it is complementary to the target gene, it directly targets the cleavage of the mRNA; when it is not complete with the target gene When fully integrated, it plays a role in regulating gene expression.
  • the invention further provides a method of expressing a protein of interest using an expression system, which is the expression system described above.
  • the method comprises: (1) causing the eighth nucleic acid molecule to comprise a nucleic acid sequence encoding a first protein of interest, the fifth nucleic acid molecule comprising a nucleic acid sequence encoding a second protein of interest; 2) expressing the first protein of interest or the second protein of interest by one of: inhibiting expression of the first regulatory protein by the ninth nucleic acid molecule to express the first protein of interest; A tenth nucleic acid molecule inhibits expression of the second regulatory protein to express the second protein of interest.
  • the second regulatory protein-mediated inhibition of the first promoter is released, and the second protein of interest is co-operating with the cell-specific promoter and the first promoter.
  • the first protein of interest is efficiently activated in a specific cell by a combination of a cell-specific promoter and a second promoter. expression.
  • the HEK293 (293-H) cell line was purchased from Invitrogen, the human hepatoma cell line HepG2, the normal liver cell Chang was purchased from ATCC, the human liver cancer cell Huh7 was purchased from the North Natron, and Hepa1-6 was purchased from the ATCC.
  • the cells were cultured in Dulbecco's Modified Eagle's Medium, 4.5 g/L glucose, 0.045 unit/mL penicillin and 0.045 g/mL streptomycin, and 10% FBS (purchased from Invitrogen) at 37 °C. , humidity 100%, CO 2 concentration 5%.
  • a 24-well plate (Falcon) was taken, and 0.5 mL of a high glucose DMEM medium containing about 7.5 ⁇ 10 4 HEK293 cells was added to each well for 24 hours. Prior to transfection, change the medium and add new DMEM complete medium. Transfection experiments were performed using Attractene Transfection Reagent (Qiagen) or Lipofectamine LTX (Life Technologies). When Attractene was used, the DNA was mixed by volume, and 1.5 ⁇ L of Attractene was added, and allowed to stand at room temperature for 15 minutes, and then added to the cells.
  • pDT7004 pUBI-linker-NOS contains a maize ubiquitin promoter (UBI), downstream of the NOS terminator, and no coding protein sequence between UBI and NOS. This application uses pDT7004 to separate plasmid DNA from different experimental groups. After transfection, culture was carried out for 48 hours for flow cytometry analysis or cytokine detection related experiments.
  • AFP promoter truncation experiment-related plasmid construction AFP enhancer sequence and AFP promoter sequence were directly PCR-derived from the genome of liver cancer cell line by designing PCR primers. Point mutations in the AFP promoter sequence were obtained by overlay PCR. The AFP enhancer and AFP promoter sequences were constructed in the entry clone sequence, and the recognition sequences of LR enzyme were designed at both ends. The specific promoter was constructed on the same vector as the activation gene Gal4VP16 by LR. The targeting sequence of the microRNA was inserted into the CMV-EYFP vector (XbaI/SalI) by primer annealing. The system level component library is built by Golden Gate.
  • Type IIs restriction enzymes are a special class of restriction enzymes, and the most commonly used type II restriction enzymes in molecular biology (recognition sites are palindromes).
  • the symmetric sequence, the cleavage site overlaps with the recognition sequence, is a non-palindromic symmetrical sequence, and the cleavage site is located outside the recognition site. Therefore, when the two fragments digested by the type IIs restriction endonuclease are ligated, the original recognition site will no longer exist, and will not be cleaved again in the subsequent restriction enzyme ligation reaction. The effect of welding death.
  • the Golden Gate cloning technique utilizes this property of the type IIs restriction enzyme to artificially design different sequences of cleavage sites outside the recognition sequences of these enzymes, and then use the same restriction enzyme to generate different viscous ends, thereby
  • the ability to assemble multiple fragments overcomes the deficiencies of traditional multi-fragment assembly requiring the simultaneous use of multiple different restriction enzymes. Compared with the traditional enzyme-cutting technology, the biggest feature of this technology is that it does not introduce any extra "scar", thus achieving the seamless connection of DNA fragments.
  • the type IIs restriction endonuclease used in the first-stage element library in this example is Esp3I.
  • the system's secondary logic line library was also constructed by Golden Gate, using the Type IIs restriction endonuclease as BsaI.
  • Logic ViraPower TM Adenoviral Gateway inserted into the adenoviral vector sequence in TM Expression Kit by Gateway embodiment; inserted into the Adeno-X Adenoviral System adenoviral vector sequences 3 clones described by Gibson.
  • the Gateway technology is based on the well-studied ⁇ phage site-specific recombination system (attB ⁇ attP ⁇ attL ⁇ attR) that has been studied. The two reactions of BP and LR constitute the Gateway technology.
  • the BP reaction uses an attB DNA fragment or a recombination reaction between an expression clone and an attP donor vector to create an entry clone.
  • the LR reaction is a recombination reaction between an attL entry clone and an attR destination vector.
  • the LR reaction is used to transfer the sequence of interest to one or more destination vectors in a parallel reaction.
  • the inventors employed an LR reaction.
  • Gibson assembly technology also known as "Gibson thermostatic one-step assembly method” is a DNA assembly method created by Gibson et al. at the J. CraigVenter Institute in the United States, using the synergistic effect of T5 exonuclease, DNA polymerase and ligase.
  • a plurality of DNA fragments with overlapping sequences are assembled in vitro.
  • the T5 exonuclease has 5' ⁇ 3' exonuclease activity, and can cleave a DNA fragment having an overlapping region from the 5' end to generate a 3' overhanging end, and then the overlapping sequence of the single-stranded DNA is specific at 50 °C. Annealing (at this time T5 exonuclease is gradually inactivated), and finally DNA polymerase and Taq ligase repair the double-stranded DNA, thereby forming a complete DNA molecule for seamless splicing.
  • the quantitative PCR primers were qAFP for: CAAAGCTGAAAATGCAGTTGAATG (SEQ ID NO: 15); qAFP rev: TTCCCCATCCTGCAGACAATCC (SEQ ID NO: 16); GAPDH for: AGAAGGCTGGGGCTCATTTG (SEQ ID NO: 17); GAPDH rev: AGGGGCCATCCACAGTCTTC (SEQ ID NO: 18) ). Fluorescent dye for PowerUp TM Green Master Mix (A25741 Thermo Fisher).
  • the high-purity endotoxin-free recombinant adenovirus plasmid was extracted: PacI was linearized and transfected into HEK293 cells, and transfected overnight to complete medium. The first generation was cultured for about one week. The medium was added according to the cell growth state, and then the cells were collected and 1 mL. The culture solution was placed in a 15 mL centrifuge tube, frozen and thawed three times at 37 ° C in liquid nitrogen, and centrifuged at 2000 rpm for 5 minutes. The supernatant was taken as the original solution of the virus solution.
  • a large number of virus amplifications (10 15 cm plates) were performed after repeated amplification of the virus for three consecutive generations, and then the virus was purified by CsCl density gradient centrifugation-dialysis.
  • the CsCl gradient was prepared by adding 2 mL of a CsCl solution having a density of 1.4 g/mL, and then slowly adding 3 mL of a CsCl solution having a density of 1.3 g/mL, and then adding 5 mL of the virus suspension. Centrifuge at 20000 rpm for 4 hours at 4 °C.
  • Viral bands with densities between 1.3 g/mL and 1.4 g/mL were collected into dialysis bags (the dialysis bags were boiled with 10 mM EDTA-Na 2 for 10 minutes before use).
  • dialysis buffer 50 g sucrose, 10 mL 1 M Tris-HCl, pH 8.0, 2 mL 1 M MgCl 2 to 1 L
  • the mixture was dialyzed overnight at 4 ° C, and the dialysate was changed twice.
  • the virus was collected and the virus titer was determined.
  • the virus titer was detected by using a viral DNA quantitative method after infecting cells. About 2 x 10 6 HeLa cells/well were seeded onto 6-well tissue culture plates.
  • the cell coverage reached 90-100%, and 5 ⁇ L of the virus stock solution was diluted with 500 ⁇ L of serum-free medium to infect the cells and set up two duplicate wells. Incubate at 37 ° C for 3 to 6 hours. The medium was removed and washed twice with 1 mL PBS/well. The cells were lysed by the addition of 500 ⁇ L of freshly prepared NP-40 lysis buffer (0.65% NP-40 substitute (Calbiochem, Billerica, MA, USA), 150 mM NaCl, 10 mM Tris, pH 8.0).
  • Standard curve was prepared: The PCR L2 sequence of interest was constructed on a T vector, and the plasmid was diluted to 1 ⁇ 10 9 to 1 ⁇ 10 2 copy / ⁇ L as a standard sequence.
  • the number of cells was measured by MTS method: 20 ⁇ L of MTS/PMS mixture (Promega) was added to each well of the cells to be cultured in a 96-well plate, and after mixing, the culture was continued at 37 ° C for 1 to 4 hours. Shake the plate for 10 seconds after color development and mix the colors. The absorbance (OD value) at a wavelength of 490 nm was measured using a microplate reader. A standard curve is drawn to detect the number of cells in the target cell.
  • the biological activity of cytokines can be detected by proliferation of factor-dependent cells.
  • the biological activities of IL-2, mGM-CSF, and hGM-CSF were detected using CTLL-2, FDC-P1, and TF-1 cell lines, respectively.
  • the cells to be tested for factor-dependent were collected, and 1 ⁇ 10 4 cells to be tested were added to a 96-well plate, and then a sample containing the cytokine to be tested was added, and cultured at 37° C. for 24 hours or 48 hours, and cell proliferation was detected using the MTS method. Dose-proliferation standard curve was drawn and the activity level of the corresponding cytokine in the sample was calculated by comparison.
  • Co-immunoprecipitation The cells to be tested are collected, and the cells are lysed using an appropriate amount of protein lysate (Biyuntian Co., Ltd.), and the cell debris is removed by centrifugation. A small amount of lysate was taken for Western blot analysis. The remaining lysate was added to 10-30 ⁇ L of protein A/G-agarose beads (Thermo Fisher Scientific) coupled with the corresponding tag antibody, and added to the cell lysate, and slowly shaken at 4 °C. overnight. After incubation, the supernatant was removed by centrifugation.
  • protein lysate Boyuntian Co., Ltd.
  • the experimental animals were purchased from Huakang Kang or Weitong Lihua Company, and the tumor model was 6-week old male or female mice, which were kept in the environment of SPF.
  • the human tumor model was constructed using nude mice. 1 ⁇ 10 7 HepG2 human hepatoma cells mixed with Matrigel (BD) were injected subcutaneously, inoculated into the right abdomen of nude mice, and the size of the subcutaneous tumor mass was measured by vernier calipers every three days.
  • the murine tumor model was constructed using C57BL/6J wild type mice, and 1 ⁇ 10 6 Hepa 1-6 mouse liver cancer cells were subcutaneously inoculated into the right abdomen of the mice, and the size of the subcutaneous tumor pieces was measured by vernier calipers every three days.
  • oncolytic viruses are a very effective means of treating cancer, and oncolytic adenovirus has carried out many clinical studies.
  • the oncolytic adenovirus has a relatively high proportion of leakage, so how to improve the safety of oncolytic adenovirus in the clinical stage is an important issue.
  • the regulation of most oncolytic adenoviruses relies on a single biomarker, such as a tumor-specific promoter that regulates the expression of the key gene E1 of adenoviral replication.
  • a single biomarker has a relatively high probability of leakage.
  • the inventors designed a dual suppression switch system to respond to a variety of biomarkers, and at various levels regulate the expression of the adenovirus E1A gene, thereby improving the recognition specificity of the oncolytic adenovirus and thereby enhancing safety.
  • the dual suppression switch system has the following features:
  • the tumor specific promoter (pC) regulates the total activator of the dual suppression switch system.
  • the activator acts on the activator response promoter (pAct) to regulate the expression of downstream genes.
  • One side of the double suppression system expresses the E1A gene associated with adenovirus packaging, the effector (Effector) and the inhibitor a (Rep-a, repressor a), and the targeting of microRNA a (miRNA a) highly expressed in non-tumor cells. sequence.
  • the promoter on this side has a recognition sequence (Tb, target of repressor b) expressed on the other side and a targeting sequence in response to the highly expressed microRNA b (miRNA b) in the tumor cells.
  • the other side of the double suppression system expresses the repressor b (Rep-b, repressor b).
  • the promoter on this side has a recognition sequence (Ta, target of repressor a) on the other side.
  • pC initiates the expression of the whole system. Because of the high expression of miRNA b and the low expression of miRNA a in tumor cells, the inhibitor b is degraded by RNA interference, which inhibits the inhibitory effect of suppressor b on the other side. .
  • the high expression of the inhibitor a can further inhibit the expression of the inhibitor b, and the double suppression switch system can be rapidly and efficiently flipped to the state of E1A expression, and the expression of the packaging and effector factors of the adenovirus is initiated.
  • the double-inhibitor switch enters non-tumor cells, the combination of micro-RNA markers and logic lines through tumor-specific promoters, microRNA markers and logic lines cannot express E1A and effector factors, effectively closing the packaging process of adenovirus, ensuring The specificity and safety of this logic. Therefore, such switches can distinguish between target and non-target cell types at the transcriptional and post-transcriptional levels in response to a variety of input signals.
  • the system designed by the inventor can distinguish the target cells from multiple levels and levels, and can effectively improve the safety of the adenovirus.
  • AFP liver cancer-specific alpha-fetoprotein
  • Alpha-fetoprotein ( ⁇ FP or AFP) is mainly synthesized in fetal liver. There is no AFP expression in normal adults, but when hepatocytes become cancerous, AFP is re-expressed, and AFP is in serum as the disease progresses. The amount will increase dramatically.
  • alpha-fetoprotein is a specific clinical indicator for the diagnosis of primary liver cancer. Therefore, in the present example, the inventors selected the alpha-fetoprotein promoter as a promoter marker for distinguishing between liver cancer cells and non-hepatoma cells.
  • RNA of Chang, HepG2, Huh7, PLC, Hep3B and Hepa1-6 cells was extracted as shown in FIG. The expression level of the AFP gene in these cells was examined by quantitative PCR.
  • Chang is a normal human liver cell
  • HepG2, Huh7, PLC, and Hep3B are human liver cancer cells.
  • Hepa1-6 is a liver cancer cell of a mouse.
  • the human AFP gene is highly expressed in the HepG2 and Huh7 cell lines and is low in the PLC and Hep3B cell lines. A slight expression was also detected in the mouse hepatoma cell Hepa1-6.
  • the currently widely used AFP promoter consists of an enhancer and a promoter.
  • the enhancer contains the sequences DA (domain A) and DB (domain B) of the two-stage activation promoter.
  • the inventors mutated the G at position -119 in the promoter region to A.
  • the inventors performed different truncation experiments on the enhancer region.
  • AFPI contains an enhancer sequence of 1.8K of AFP (sequence is shown as SEQ ID NO: 21); AFPII contains activation region A (DA), activation region B (DB) and two activation regions.
  • Inter-sequence (sequence as shown in SEQ ID NO: 22); AFPIII contains only activation region A (DA) and activation region B (DB) (sequence as shown in SEQ ID NO: 23); AFPIV contains only activation region A (DA) (sequence as shown in SEQ ID NO: 24); AFPV contains only activation region B (DB) (sequence as shown in SEQ ID NO: 25).
  • the expression efficiency and specificity of the AFP promoter were detected by transient transfer of these five AFP promoter reporter system plasmids into normal hepatocytes Chang and hepatoma cells HepG2.
  • the five AFP promoters have high expression levels in liver cancer cell lines, even exceeding the commonly expressed CMV promoter. From the specificity analysis, in addition to the AFPII reporter gene in Chang, there was no obvious leakage of other promoters. Considering the length of the inserted sequence, the efficiency and specificity of the promoter. In the subsequent experiments, the inventors selected the AFP III promoter to regulate the inventors' dual suppression switch.
  • microRNA microRNA
  • microRNA microRNA
  • Table 1 there are many highly expressed microRNAs in different cancer cells.
  • the inventors used miR199a as a microRNA marker to characterize the high expression of normal hepatocytes in normal hepatocytes, and accordingly the inventors used miR21 and miR122 as microRNA markers to characterize the high expression of hepatoma cells in hepatoma cells. Things.
  • the inventor designed and constructed a microRNA fluorescent plasmid reporting system, as shown in Figure 7.
  • the inventors inserted the target sequence of the microRNA into the 3' untranslated region of the frequently expressed EYFP and the other frequently expressed EBFP as a control fluorescence.
  • a total of different cell lines were transferred to detect the expression of the target microRNA in the target cell line.
  • the results of the experiment are shown in Fig. 8.
  • miR199a is highly expressed in normal hepatocytes; while the expression level of miR21 in hepatoma cell lines is significantly increased, which can be distinguished as normal in the double suppression switch system designed by the inventors.
  • An effective microRNA marker for cells and liver cancer cells is highly expressed in normal hepatocytes.
  • Double suppression switch can effectively respond to microRNA input signals and achieve stable function in different cell lines.
  • the inventors Since the packaging capacity of the oncolytic adenovirus is limited, only 8K foreign genes can be packaged at most, so the inventors have selected short and effective suppressing elements as much as possible in the construction of this embodiment.
  • the inventors selected LacI and tetR-KRAB. As shown in Figure 9, the inventors constructed two logical lines: whether the EYFP gene is co-expressed on the tetR-KRAB side. Switch I (SI) does not express EYFP, and Switch II (SII) expresses EYFP. The inventors transiently transferred the two switches into HEK293, and simultaneously shRNA-FF4 and shRNA-FF5. As shown in Figure 10, the inventors' two switches can be efficiently flipped under different input signals.
  • SI expressed higher levels on the LacI side, but the level of leakage on the tetR-KRAB side was also higher than that of SII.
  • the inventors chose the SI system as the backbone to construct the oncolytic adenovirus carrying the different effectors of the inventors.
  • the inventors examined the control effects of different activators on the bidirectional suppression switch. As shown in Figure 11, the inventors examined the regulation efficiency of Gal4VP16, Gal4esn, dCas9-VP64 and rtTA. The experimental results show that the gene line can respond effectively to different activators, so the gene line has good stability.
  • a marker element that distinguishes a normal cell from a cancer cell such as a tumor-specific promoter, a regulatory element related to a logical line, and a recognition sequence that distinguishes a microRNA of a cancer cell, is constructed on a primary vector plasmid.
  • a primary vector plasmid Form a library of primary components.
  • the recognition sites of EI a restriction enzyme with different recognition sites and cleavage sites such as Esp3I and BsaI
  • EI a restriction enzyme with different recognition sites and cleavage sites such as Esp3I and BsaI
  • the inventors designed an in vitro cell killing assay for oncolytic adenovirus. Different cell lines are infected with different infection numbers. Chang is a normal liver cell, and Huh7 and HepG2 are human liver cancer cell lines. The viability of the cells was measured by the MTS method after the virus culture for six days. As shown in Fig. 15, it was found that when the multiplicity of infection was 100, 10, 1, the liver cancer cells showed significant cytotoxicity and died a lot, and there was a slight cell death at a multiplicity of infection of 0.1.
  • the multiplicity of infection when the multiplicity of infection is 10 or less, the growth state of Chang cells is normal and there is no death. At a multiplicity of infection of 100, the cells have a slight cytotoxic response, while the cell volume becomes larger and the cells grow slower. This data indicates that the adenovirus packaged by the inventors can effectively distinguish between liver cancer cells and the growth and proliferation of normal liver cells within a specific multiplicity of infection.
  • the adenoviral vectors currently used clinically are human Ad2 or Ad5 type adenoviruses, these viruses are widely present in nature, and neutralizing antibodies that antagonize these adenoviruses already exist in the human body. Therefore, only relying on the oncolytic effect of adenovirus to treat tumors, clinical data show that the treatment effect is not good. Therefore, in the present embodiment, the inventors used the oncolytic adenovirus as a vector to carry the effector gene to the target site of the tumor to cause a systemic immune response. As shown in Figure 16, the inventors first constructed these effectors on a plasmid vector that was frequently expressed, and detected the activity of these effectors by means of a transient.
  • the inventors constructed these effector genes into an oncolytic adenovirus vector, and packaged and purified the oncolytic adenovirus carrying these effectors. After infecting the hepatoma cell HepG2, the inventors extracted the supernatant to detect the activity of the immune factor in the supernatant.
  • Fig. 17a 12-well plates were inoculated with 3 ⁇ 10 6 HepG2 cells, and Synp-EBFP and synOV-mGM-SCF were used to infect HepG2 with a multiplicity of infection of 10, respectively, at 1, 2, 3 The supernatant was collected for 4 days, and the content of GM-CSF in the supernatant was detected by an ELISA kit.
  • Figure 17b12 Orifice plate was inoculated with 3 ⁇ 10 6 HepG2 cells, and HepG2 was infected with synOV-EBFP and synOV-hIL-2 at a multiplicity of infection of 10, respectively.
  • the supernatant was collected on 1, 2, 3, and 4 days, respectively, and the supernatant was added to IL.
  • the content of IL-2 in the supernatant was detected by MTS.
  • FIG. 17c 12-well plates were seeded 3 ⁇ 10 6 HepG2 cells, respectively synOV-EBFP, synOV-anti- PD-1scFv and synOV-anti-PD-L1scFv infected at a multiplicity of infection of 10 HepG2.
  • the supernatants were collected on 1, 2, 3, and 4 days, respectively.
  • Mouse spleen cells were isolated, T cells were activated with 2 ug/mL anti-mouse CD3 antibody, and the supernatant collected above was added to the spleen cell system at a dilution ratio of 1:10.
  • the IFN- ⁇ production in splenocytes was measured by ELISA after 48 hours.
  • HepG2 and Huh7 are human hepatocarcinoma cells.
  • Hepa1-6 is a mouse hepatoma cell. After the tumor volume reaches 100 mm 3 , it is randomly divided into two groups. The treatment group is intratumorally injected with 1 ⁇ 10 9 oncolytic adenovirus, and injected with PBS. Control group.
  • the tumor in the treatment group continued to grow, and the oncolytic virus treatment group could control the growth of the tumor to some extent, but did not effectively control the tumor growth, probably because Hepa1-6 grew faster than HepG2 and Huh7, and at Hepa1-6.
  • the expression level of AFP is relatively low. Therefore, the inventors continued to treat the Hepa1-6 tumor model by using the oncolytic adenovirus carrying the immune effector factor in subsequent experiments, and achieved very effective results.
  • the therapeutic effect of the inventor's oncolytic adenovirus on a C57 mouse model with an immune system was further examined.
  • 1 ⁇ 10 6 Hepa1-6 mouse hepatoma cells were subcutaneously inoculated into the right abdomen of wild-type C57BL/6J mice. After the tumor volume reached 100 mm 3 , each group was intratumorally injected with 1 ⁇ 10.
  • 9 Express different cytokine oncolytic adenoviruses, or PBS as a negative control. The volume of the tumor after treatment was observed and the survival time of the tumor-bearing mice was analyzed by Kaplan-Meier method.
  • mice with intact immune system oncolytic adenovirus can still inhibit tumor growth and significantly prolong the survival time of tumor-bearing mice.
  • One month after the oncolytic adenovirus treatment 1 ⁇ 10 6 Hepa 1-6 mouse liver cancer cells were inoculated again in the left abdomen of the surviving mice, and it was found that the same tumor cells were inoculated in the same part of the same mouse as the primary vaccination. Department, can not grow into a tumor. This proves that in mice treated with oncolytic virus, the body produces specific immunity against tumor cells, which can resist the same tumor cells from growing again in the body.
  • Tumors of tumor-bearing mice were taken, and the lymphocyte infiltration of tumor tissues was detected by HE staining after fixation and sectioning. It was found that the lymphocyte infiltration of tumor tissues of mice treated with oncolytic adenovirus was significantly increased compared with the PBS control group, demonstrating that treatment with oncolytic adenovirus recruited more lymphocytes in tumor tissues.
  • the infiltrating lymphocytes in the tumor were isolated and purified, and the phenotype of mouse tumor infiltrating T cells was detected by flow cytometry. It was found that the proportion of Ki-67 + T cells infiltrated by tumors in mice treated with oncolytic virus was higher.
  • Tumor-infiltrating lymphocytes were added to RPMI 1640 medium containing PMA (20 ng/mL) and Ionomycin (1 ⁇ g/ml L), and cultured at 37 ° C for 4 hours in the presence of Brevedlin A. After cell fixation staining, the expression of ⁇ -interferon in CD4 + and CD8 + T cells was detected by flow cytometry. It was found that in the tumor of the oncolytic adenovirus-treated group, the expression of ⁇ -interferon was higher in T cells, which changed the immune microenvironment inside the tumor and promoted the clearance of tumor cells, as shown in Fig. 20 (a, b). Shown.
  • Oncolytic adenovirus treatment causes a systemic anti-tumor immune response
  • Mouse Hepa-1-6 tumors were inoculated subcutaneously on the left and right sides of the abdomen of C57BL/6 mice. After tumor formation, the cytokine hIL-2, mGM-CSF, anti-PD- were injected into the right tumor. 1 scFv of oncolytic virus, PBS control and AdGFP control, continued measurement of tumor growth. After 14 days of treatment, tumors on both sides of tumor-bearing mice were taken. Lymphocytes infiltrated in the tumor were isolated and purified, and the phenotype of tumor infiltrating T cells in mice was detected by flow cytometry.
  • oncolytic adenovirus The therapeutic situation of oncolytic adenovirus is abstracted into a tumor-virus-immune system, as shown in Figure 22, containing uninfected tumor cells, infected tumor cells, free virus, immune cells activated by oncolytic adenovirus antigens.
  • Five components of immune cells activated by tumor antigens are represented by S, I, V, Z V and Z T , respectively.
  • the model assumes that the growth of uninfected tumor cells conforms to the generalized logistic growth model, where ⁇ is the growth rate coefficient, K is the environmental capacity, and ⁇ is the nonlinear coefficient.
  • Uninfected tumor cells can be infected by free virus and transformed into infected tumor cells with an invasive rate of ⁇ .
  • the infected tumor cells will lyse and release more virus with a lysis rate coefficient of ⁇ and a free virus release rate coefficient of ⁇ .
  • the free virus naturally decays in the environment with a decay rate coefficient of ⁇ .
  • Z V is increased by the activation of oncolytic adenovirus and tumor cell surface antigen infected by virus.
  • the rate coefficients are c V1 and c V2 , respectively, and Z V can inhibit the infected tumor cells.
  • the rate coefficient is p V .
  • Z T is increased by the activation of antigen released by tumor cell lysis, and the rate coefficient is c T , and Z T can inhibit the infected and uninfected tumor cells with a rate coefficient of p T .
  • Z V and Z T are naturally attenuated in the environment with a rate coefficient of ⁇ .
  • the simulation plot shows the minimum tumor volume during the period when the tumor-virus system changes to the final state over time when the virus replication rate is lower and higher under different initial virus titers and initial tumor size conditions. Changes. It can be seen from the simulation results that increasing the initial titer of the virus helps to control the tumor size.
  • the appropriate initial dose can be selected in conjunction with the initial tumor size and dose limit to allow tumor clearance or supplementation with other therapies at the predicted time points, as shown in FIG.
  • the tumor size in the final state of the system during the simulation time Under the influence of the different inhibitory effects of immune cells on tumors and viruses, the tumor size in the final state of the system during the simulation time. It can be seen from the simulation results that the system exhibits several dynamic behaviors that tend to completely eliminate tumors, tumor growth to saturation, tumor volume stability, and tumor volume oscillation. In general, the stronger the inhibitory effect of immune cells on tumors, the more the system tends to clear the tumor, and the stronger the inhibitory effect of immune cells on the virus, the more the system tends to lose control of the tumor, and for the tumor to stabilize or oscillate, the system is about two The trend of immunization showed some non-monotonic changes, and the results are shown in Figure 24.

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Abstract

一种表达系统，包括:有细胞特异性启动子的第一核酸分子；编码转录激活因子的第二核酸分子；有所述转录激活因子的第一识别序列的第三核酸分子；有第一启动子和第一调控元件的第四核酸分子；编码第一调控蛋白的第五核酸分子；有所述转录激活因子的第二识别序列的第六核酸分子；有第二启动子和第二调控元件的第七核酸分子；编码第二调控蛋白的第八核酸分子；以及：被配置为条件性抑制所述第一调控蛋白的表达的第九核酸分子；被配置为条件性抑制所述第二调控蛋白的表达第十核酸分子，其中，第一调控元件适于通过结合第二调控蛋白抑制第一启动子的功能，第二调控元件适于通过结合第一调控蛋白抑制第二启动子的功能。

Description

可编程的溶瘤病毒疫苗系统及其应用
优先权信息
本申请请求2017年03月24日向中国国家知识产权局提交的、专利申请号为201710184736.2的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物医药领域，具体地，本发明涉及表达系统及其应用，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，进一步地，本发明还涉及利用表达系统表达目的蛋白的方法。
背景技术
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(Gendicine和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。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。
综合以上的研究成果可以发现，溶瘤病毒确实是治疗肿瘤的利器，但依然存在着很多的问题。
目前已知的人体腺病毒有52种，分别命名为Adl～Ad52，研究中最常用的是Ad2。腺病毒的转录模式具有十分鲜明的特点：已知的腺病毒基因组的转录单位至少有5个：E1区位于病毒基因组左侧，可再分成E1A和E1B，与细胞转化有关；E2区编码DNA结合蛋白，参与病毒的复制；E3区编码出现在宿主细胞表面的一种糖蛋白；E4区位于Ad2基因组右端，受E2区编码的DNA结合蛋白质调控；第5个转录单位在病毒感染中期合成Ad2蛋白质Ⅳ。腺病毒基因组进入细胞核后，细胞转录因子首先与E1A区上游的增强子结合，表达E1A蛋白，该蛋白的作用是调节细胞代谢，使病毒DNA更易于在细胞中复制。E1A蛋白还可以激活其他早期基因(E1B、E2A、E2B、E3和E4)的启动子，其中E2B驱动另外三个与病毒复制有关的早期基因转录单位末端蛋白前体(pTP,precursor terminal protein)、单链DNA结合蛋白(ssDBP,single-stranded DNA binding proteins)以及DNA聚合酶(DNA pol,DNA polymerase)的表达，这三个基因的表达产物紧密地结合成一个复合物，与至少三种细胞蛋白相互作用，启动病毒基因组的复制。通过对腺病毒基因组和转录模式的分析，可以做出以下的改造策略：通过调控E1A的表达来调控腺病毒在靶向细胞中的表复制包装；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。
而现有的溶瘤腺病毒的研究结果显示现有的调控方式存在过于单一，特异性差，安全性低等缺陷，同时腺病毒的改造技术方法比较繁琐。所以如何设计更加严谨安全的调控系统以及如何快速高效的实现溶瘤腺病毒的拼装是设计构建溶瘤腺病毒急需解决的重要问题。
发明内容
本发明旨在至少在一定程度上解决相关的技术问题之一。
基于发明人实验室的研究基础，发明人设计并构建了一个适用于腺病毒调控的可以响应不同微环境的基因线路。在此基因线路中，发明人利用了多水平的生物标志物。具体地，第一，利用特异性启动子作为调控基因线路的总开关，即利用特异性启动子调控总激活子的表达，进一步调控腺病毒E1A基因的表达。第二，利用microRNA的靶标序列来响应不同微环境的microRNA的表达特征作为调控基因线路的二级开关。第三，利用闭合基因线路搭建开关系统，闭合的双向抑制型开关可以更高效的响应微环境的变化，同时有效的避免 泄露。第四，移除了E1B基因，利用E1B识别P53基因的互补缺失原理进一步提高腺病毒区分肿瘤细胞和正常细胞的能力。第五，移除了E3基因，降低了溶瘤腺病毒对正常细胞的毒害作用同时提高了腺病毒载体的包装容量。
同时，2型和5型人腺病毒的基因组长度为36K，如果利用传统单一的质粒构建方式(如酶切连接等)改造腺病毒的难度很大，耗时很长。为了解决这一问题，发明人设计了针对本发明涉及的基因线路搭建的腺病毒快速构建方法：第一步构建一级元件库，将所需的元件构建到相应的质粒上。一级元件库包括三个库：抑制元件A库主要表达基因线路一侧的抑制元件，腺病毒的E1A基因和与腺病毒共表达的效应因子基因(如免疫因子或杀伤基因)；抑制元件B库主要表达基因线路另一侧的抑制基因，用于调控基因线路的有效翻转，增强开关的安全性；特异性启动子库，通过组织或肿瘤特异性的启动子调控下游基因线路的总开关。第二步，确定生物标志物和表达的效应因子，从一级元件库中挑选相应的质粒，通过Golden Gate的方法快速搭建基因线路。第三步通过Gateway的方式将基因线路整合入已经改造好的腺病毒载体上(去除E1基因，便于实现对腺病毒的人工控制；去除部分E3序列，以扩大包装容量)。
溶瘤腺病毒的另一个显著特点就是可以携带并同时表达多个基因，在本发明中，发明人同时表达了单个或多个细胞免疫相关基因：如IL-2、GM-CSF,、anti-PD-1scFv、anti-PD-L1scFv以及这些因子之间的融合蛋白等。由于携带免疫相关基因，溶瘤病毒在攻击肿瘤细胞的同时会引起系统性的免疫反应，但也存在引起免疫过激反应的风险和隐患。所以携带何种免疫反应基因，以及相应的给予多大剂量的病毒治疗，以及病毒治疗的给药方式等都强烈影响溶瘤病毒的治疗效果。在本发明中,发明人尝试利用生物信息学的方法对溶瘤腺病毒感染靶向细胞的过程进行建模，研究溶瘤腺病毒杀伤肿瘤细胞的特性，尽量地提高了溶瘤腺病毒的治疗效果。
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，所述表达系统包括：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；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；以及选自下列的 至少之一：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；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。利用根据本申请实施例的表达系统，能够实现目的基因在特定细胞环境中的特异性表达，对目的基因的特异性表达实现了双向调控，调控力度大而有效，且特异性强。
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：
根据本发明的实施例，所述细胞特异性启动子为肿瘤细胞特异性启动子，所述肿瘤细胞特异性启动子为选自甲胎蛋白特异性启动子，Survivin基因启动子,人端粒酶逆转录酶基因启动子,胆囊收缩素A受体基因启动子，癌胚抗原启动子，原癌基因人类表皮生长因子受体2启动子，前列腺素内氧化酶还原酶2启动子，趋化因子受体-4，E2F-1基因启动子，黏蛋白启动子，前列腺特异抗原，人酪氨酸酶相关蛋白1，酪氨酸酶启动子至少之一。在上述肿瘤细胞特异性启动子的调控下，本申请实施例的表达系统能够在特异肿瘤细胞的微环境中启动，表达系统对基因表达调控的特异性进一步增强。
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。
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。
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。
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)、KRAB、tetR-KRAB、dCas9-KRAB至少之一。
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)以及dCas9的靶标序列至少之一。
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。
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。
根据本发明的实施例，所述第五核酸分子和第九核酸分子的至少之一进一步包括编码目的蛋白的序列。利用根据本申请实施例的表达系统，可实现目的蛋白在特异细胞微环境中的特异性表达，实现了对目的蛋白表达的双向调控。根据本发明的具体实施例，本申请所述的表达系统对目的蛋白的表达调控的特异性和力度均显著提高。
根据本发明的实施例，所述第五核酸分子包括编码所述目的蛋白的序列，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。任选地，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，进而促进对肿瘤细胞等特异性细胞的免疫杀伤。
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。
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。
根据本发明的实施例，所述目标蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目标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。目标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，目标蛋白与第一调控蛋白分开，目标蛋白和第一调控蛋白相互独立地发挥功能。
根据本发明的实施例，所述第九核酸分子与所述第十核酸分子分别独立地通过下列方式抑制所述第一调控蛋白或所述第二调控蛋白的表达：借助RNA干扰。其中，microRNA为不同细胞微环境表达的特异性microRNA，第九或第十核酸分子为microRNA的特异性靶标序列，进而通过RNA干扰(RNAi),可实现在特异性微环境中表达的microRNA对靶标序列的特异性作用，进而实现对第一调控蛋白或第二调控蛋白表达的特异性调控。根据本发明的实施例，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。进而，第一调控蛋白在异常细胞中表达，在正常细胞中不表达或低表达，而第二调控蛋白在正常细胞中表达，在异常细胞中不表达或低表达。
根据本发明具体的实施例，所述第一microRNA包括选自下列的至少之一：miR199a、miR95、miR125、miR125b、Let-7、miR143、miR145以及miR200C。上述microRNA在正常肝细胞中高表达。
根据本发明的具体示例，所述第二microRNA包括选自下列的至少之一：miR21、miR223、 miR224、miR221、miR18、miR214、miR146a以及miR1792。上述第二microRNA为肝癌细胞(HepG2、Huh7、PLC)特异性表达的microRNA。进而，第一调控蛋白在肝癌细胞中表达，在正常细胞中不表达或低表达，而第二调控蛋白在正常细胞中表达，在肝癌细胞中不表达或低表达。
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可选的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可选的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，在适宜的微环境，如细胞中，实现对目的基因的特异性表达的调控。
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适宜微环境中的功能的发挥即可。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：
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。
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。
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。
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。
根据本发明的具体实施例，所述第一表达载体包括：从5’端到3’端依次为BsaI，AFP III，Gal4VP16以及BsaI(BsaI-AFP III-Gal4VP16-BsaI)。
根据本发明的实施例，所述第一表达载体携带具有SEQ ID NO：1所示核苷酸序列的核酸。
Figure PCTCN2017096043-appb-000001
Figure PCTCN2017096043-appb-000002
Figure PCTCN2017096043-appb-000003
根据本发明的具体实施例，所述第二表达载体包括：从5’到3’依次为BsaI，5×UAS，tetO，miniCMV，tetO，E1A，2A，免疫效应因子(Effector)，LacI，microRNA199a特异识别序列(target site)以及BsaI(BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-Effector-LacI-miR199a target site-BsaI)。根据本发明的具体示例，effector具体指IL-2、hGM-CSF、mGM-CSF、anti-PD-1scFv、anti-PD-L1scFv、IL-2融合anti-PD-1scfv、hGM-CSF融合anti-PD-1scfv、mGM-CSF融合anti-PD-1scfv、IL-2融合anti-PD-L1scfv、hGM-CSF融合anti-PD-L1scfv、mGM-CSF融合anti-PD-L1scfv。
根据本发明的实施例，所述第二表达载体携带具有SEQ ID NO：2～7任一项所示核苷酸序列的核酸。
Figure PCTCN2017096043-appb-000004
Figure PCTCN2017096043-appb-000005
Figure PCTCN2017096043-appb-000006
Figure PCTCN2017096043-appb-000007
Figure PCTCN2017096043-appb-000008
Figure PCTCN2017096043-appb-000009
Figure PCTCN2017096043-appb-000010
Figure PCTCN2017096043-appb-000011
Figure PCTCN2017096043-appb-000012
Figure PCTCN2017096043-appb-000013
Figure PCTCN2017096043-appb-000014
Figure PCTCN2017096043-appb-000015
其中，SEQ ID NO：2是BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-EBFP-2A-LacI-miR199a target site-BsaI的序列；
SEQ ID NO：3是BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-hIL-2-2A-LacI-miR199a target site-BsaI的序列；
SEQ ID NO：4是BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-hGM-CSF-2A-LacI-miR199a target site-BsaI的序列；
SEQ ID NO：5是BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-mGM-CSF-2A-LacI-miR199a target site-BsaI的序列；
SEQ ID NO：6是BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-anti-PD-1scFv-2A-LacI-miR199a target site-BsaI的序列；
SEQ ID NO：7是BsaI-5×UAS-tetO-miniCMV-tetO-E1A-2A-anti-PD-L1scFv-2A-LacI-miR199a target site-BsaI的序列。
根据本发明的具体实施例，所述第三表达载体包括：从5’到3’依次为BsaI，5×UAS，LacO，miniCMV，LacO，tetR-KRAB，microRNA21特异识别序列(target site)以及BsaI，
(BsaI-5×UAS-LacO-miniCMV-LacO-tetR-KRAB,-microRNA21target site-BsaI)。
根据本发明的实施例，所述第三表达载体携带具有SEQ ID NO：8所示核苷酸序列的核酸。
Figure PCTCN2017096043-appb-000016
根据本发明的具体实施例，所述腺病毒包括：从5’到3’依次是第一反向末端重复序列(inverted terminal repeat ITR),包装信号(package signal)，AFPIII，Gal4VP16，5×UAS，tetO，miniCMV，tetO，E1A，2A，Effector，2A，LacI，miR199a target site，5×UAS，LacO，miniCMV，LacO，tetR-KRAB，miR21target site,腺病毒E2基因区，E3基因区(去除了28922-30801区域)，E4基因区，第二反向末端重复序列(ITR)。发明人将上述腺病毒注入肿瘤小鼠模型，可显著抑制小鼠肿瘤的生长。上述腺病毒可作为一种安全有效的溶瘤病毒疫苗，实现对相关肿瘤的安全有效地特异性杀伤。
根据本发明的实施例，所述腺病毒载体携带具有SEQ ID NO：9～14所示核苷酸序列的核酸。
Figure PCTCN2017096043-appb-000017
Figure PCTCN2017096043-appb-000018
Figure PCTCN2017096043-appb-000019
Figure PCTCN2017096043-appb-000020
Figure PCTCN2017096043-appb-000021
Figure PCTCN2017096043-appb-000022
Figure PCTCN2017096043-appb-000023
Figure PCTCN2017096043-appb-000024
Figure PCTCN2017096043-appb-000025
Figure PCTCN2017096043-appb-000026
Figure PCTCN2017096043-appb-000027
Figure PCTCN2017096043-appb-000028
Figure PCTCN2017096043-appb-000029
Figure PCTCN2017096043-appb-000030
Figure PCTCN2017096043-appb-000031
Figure PCTCN2017096043-appb-000032
Figure PCTCN2017096043-appb-000033
Figure PCTCN2017096043-appb-000034
Figure PCTCN2017096043-appb-000035
Figure PCTCN2017096043-appb-000036
Figure PCTCN2017096043-appb-000037
Figure PCTCN2017096043-appb-000038
Figure PCTCN2017096043-appb-000039
其中，SEQ ID NO：9是腺病毒载体携带的带有EBFP的核酸的序列；
SEQ ID NO：10是腺病毒载体携带的带有hIL-2的核酸的序列；
SEQ ID NO：11是腺病毒载体携带的带有hGMCSF的核酸的序列；
SEQ ID NO：12是腺病毒载体携带的带有mGMCSF的核酸的序列；
SEQ ID NO：13是腺病毒载体携带的带有anti-PD-1scFv的核酸的序列；
SEQ ID NO：14是腺病毒载体携带的带有anti-PD-L1scFv的核酸的序列。
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的方式将逻辑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。
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，所述肿瘤细胞特异性启动子为甲胎蛋白特异性启动子；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，所述第一识别序列为5×UAS；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，并且所述复制蛋白与效应因子之间通过可切割的连接肽连接的，所述目标蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目标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，所述第二识别序列为5×UAS；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。利用根据本申请实施例的重组病毒，在甲胎蛋白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、目标蛋白在肿瘤细胞中特异性表达，第二调控蛋白tetR-KRAB在肿瘤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、目标蛋白在第一启动子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，可实现蛋白在肿瘤细胞中更加特异性的表达(如目标蛋白LacI)或不表达(如tetR-KRAB)，在肿瘤细胞中的表达或不表达效率和特异性高。
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：
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。
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。
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。
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，攻击异种细胞，如肿瘤细胞，安全性高，特异性强。
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：
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。
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，所述药物用于治疗癌症。本申请所述的表达系统能够实现目的蛋白在肿瘤细胞中特异性表达，根据本申请实施例的药物对癌症的治疗效果更加有效、特异性更强、安全性更高。
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述癌症包括肝癌、肺癌、结直肠癌，黑色素瘤、乳腺癌或前列腺癌。发明人发现，根据本发明实施例的药物对肝癌、肺癌、结直肠癌，黑色素瘤、乳腺癌或前列腺癌的治疗效果更加显著。
在本发明的第五方面，本发明提出了一种利用表达系统表达目的蛋白的方法，所述表达系统为前面所述的表达系统。根据本发明的实施例，所述方法包括：(1)使所述第五核酸分子包含编码所述目的蛋白的核酸序列；(2)使所述第十核酸分子抑制所述第二调控蛋白的表达，以便表达所述目的蛋白。利用根据本发明实施例的上述表达目的蛋白的方法，第二调控蛋白介导的对第一启动子的抑制机制解除，目的蛋白在细胞特异性启动子和第一启动子的共同作用下，在特异细胞中高效表达。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述表达是在细胞中进行的。细胞可以提供目的蛋白表达的微环境，目的蛋白在细胞中的表达更加高效。
根据本发明的实施例，所述第十核酸分子含有被第二microRNA特异性识别的核酸序列，在步骤(2)中包括使所述第二microRNA与所述第十核酸分子接触。
在本发明的第六方面，本发明提出了一种利用表达系统表达目的蛋白的方法，所述表达系统为前面所述的表达系统。根据本发明的实施例，所述方法包括：(1)使所述第八核酸分子包含编码第一目的蛋白的核酸序列，使所述第五核酸分子包含编码第二目的蛋白的核酸序列；(2)通过下列之一表达所述第一目的蛋白或所述第二目的蛋白：使所述第九核酸分子抑制所述第一调控蛋白的表达，以便表达所述第一目的蛋白；使所述第十核酸分子抑制所述第二调控蛋白的表达，以便表达所述第二目的蛋白。利用根据本发明实施例的上述表达目的蛋白的方法，第二调控蛋白介导的对第一启动子的抑制机制解除，第二目的蛋白在细胞特异性启动子和第一启动子的共同作用下，在特异细胞中高效表达或第一调控蛋白介导的对第二启动子的抑制机制解除，第一目的蛋白在细胞特异性启动子和第二启动子的共同作用下，在特异细胞中高效表达。
需要说明的是，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。
附图说明
图1Gateway系统腺病毒原始结构示意图；
图2Golden Gate系统腺病毒原始结构示意图；
图3是根据本发明实施例的双抑制型开关系统；
图4是根据本发明实施例的利用荧光定量PCR的方法检测hAFP基因在细胞系Chang、 HepG2、Huh7、Hep3B、PLC和Hepa1-6中的表达水平；
图5是根据本发明实施例的改造人的AFP基因的启动子；
图6是根据本发明实施例的将改造后的AFP启动子的报告系统瞬转到Chang和HepG2；
图7是根据本发明实施例的microRNA检测系统示意图；
图8是根据本发明实施例的microRNA标志物在不同细胞系中存在显著地差异表达；
图9是根据本发明实施例的基于LacI和tetR-KRAB基因的双抑制开关示意图；
图10是根据本发明实施例的双抑制开关响应人工合成的shRNA信号实现有效翻转；
图11是根据本发明实施例的双抑制型开关有效响应各种激活因子；
图12是根据本发明实施例的CGGA(Cascade Golden-Gate and Gateway/Gibson Assemble method)第一步；
图13是根据本发明实施例的CGGA(Cascade Golden-Gate and Gateway/Gibson Assemble method)第二步；
图14是根据本发明实施例的CGGA(Cascade Golden-Gate and Gateway/Gibson Assemble method)第三步；
图15是根据本发明实施例的细胞水平检测病毒作用；
图16是根据本发明实施例的体外检测细胞因子活性(质粒载体表达细胞因子)；
图17是根据本发明实施例的体外检测细胞因子活性(病毒载体表达细胞因子)
图18是根据本发明实施例的利用溶瘤腺病毒治疗荷载HepG2，Huh7和Hepa1-6的裸鼠模型；
图19是根据本发明实施例的利用溶瘤腺病毒治疗荷载Hepa1-6的C57小鼠模型；
图20是根据本发明实施例的溶瘤病毒治疗促进肿瘤浸润的T细胞γ干扰素和Ki67标志物的表达；
图21是根据本发明实施例的小鼠肝癌细胞Hepa1-6对治疗后小鼠再接种的挑战实验；
图22是根据本发明实施例的肿瘤-病毒-免疫系统模型；
图23是根据本发明实施例的肿瘤-病毒系统仿真不同初始肿瘤大小、初始病毒滴度与病毒复制速度条件下最小肿瘤体积在不同的初始病毒滴度与初始肿瘤大小条件下，病毒复制速度较低与较高时，肿瘤-病毒系统随时间变化至终状态的过程中，肿瘤体积的最小值的变化情况；以及
图24是根据本发明实施例的肿瘤-病毒-免疫系统仿真免疫细胞对肿瘤与病毒不同的抑制作用强度影响下终状态肿瘤体积。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
表达系统
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，所述表达系统包括：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；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；以及选自下列的至少之一：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；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。利用根据本申请实施例的表达系统，能够实现目的基因在特定细胞环境中的特异性表达，对目的基因的特异性表达实现了双向调控，调控力度大而有效，且特异性强。
根据本发明的实施例，所述细胞特异性启动子为肿瘤细胞特异性启动子，所述肿瘤细胞特异性启动子为选自甲胎蛋白特异性启动子(AFP)，Survivin基因启动子(SUR),人端粒酶逆转录酶(hTERT)基因启动子,胆囊收缩素A受体基因启动子(CCKAR promoter)，癌胚抗原启动子(CEA promoter)，原癌基因人类表皮生长因子受体2启动子(human epidermal growth factor receptor-2,HER2)，前列腺素内氧化酶还原酶2启动子(Cyclooxygenase2,COX2)，趋化因子受体-4(chemokine receptor-4,CXCR4)，E2F-1基因启动子(E2F-1promoter)，黏蛋白启动子(Mucin1MUC1)，前列腺特异抗原(Prostate-specific antigen PSA)，人酪氨酸酶相关蛋白1(tyrosine related protein1TRP1)，酪氨酸酶启动子(tyrosinase Tyr promoter)至少之一。在上述肿瘤细胞特异性启动子的调控下，本申请实施例的表达系统能够在特异肿瘤细胞的微环境中启动，表达系统对基因表达调控的特异性进一步增强。
根据本发明的实施例，所述转录激活因子为选自Gal4VP16、Gal4esn、Gal4VP64、dCas9-VP16、dCas9-VP64、dCas9-VPR、dCas9-VTR以及rtTA至少之一。酵母Gal4-UAS基 因表达系统是目前了解最透彻的真核细胞转录调节系统之一，Gal4基因在酵母(Saccharomyces cerevisiae)中编码转录激活蛋白。Gal4能识别基因启动子的上游激活序列(UAS)的17bp长的一段序列：5'-CGGRNNRCYNYNYNCNCCG-3'(R表示嘌呤，Y表示嘧啶，N表示任意脱氧核苷酸)。Gal4与Gal80作用，GAL80可结合半乳糖代谢产物。Gal4具有两个结构域：DNA结合结构域(N末端结构域)和激活结构域(C末端结构域)。其具有一个锌簇结构域(Zinc cluster)，具体为Zn(2)-Cys(6)双核簇结构域。其起作用的形式常为同源二聚体。UAS序列中与GAL4蛋白结合的核心序列是一段4个拷贝串联的重复序列，每个拷贝包含17个碱基对，序列5‘-CGGAGTACTGTCGTGGG-3’。Gal4存在于哺乳动物细胞内，并且只有在识别UAS时才能激活目标基因，启动下游基因转录，具有良好的特异性和可诱导性。Gal4的蛋白激活域可以替换为单纯疱疹病毒VP16蛋白的激活结构域或者4个拷贝的VP16即VP64，这样形成的融合转录因子在保持Gal4蛋白结合特异性的同时增加了Gal4蛋白的转录激活活性。四环素诱导系统是源于细菌的用于真核细胞转录激活系统。包括四环素响应元件和四环素诱导蛋白。四环素响应元件(Tet Response Element TRE)包含7个重复的19个核苷酸的四环素操纵子序列5’-TCCCTATCAGTGATAGAGA-3’，可以被四环素诱导蛋白识别。四环素诱导系统包括四环素诱导抑制系统和四环素诱导激活系统。四环素诱导抑制系统包含四环素响应元件和四环素抑制子(tetracycline repressor tetR),当没有四环素不存在时，抑制子与响应元件结合激活下有基因的表达；当有四环素存在时，抑制子与四环素结合，下游基因的表达被抑制。四环素诱导激活系统包含四环素响应元件和反向四环素调控的顺式激活子(reverse tetracycline-controlled trans a ctivator rtTA)。当四环素不存在时，rtTA无法与TRE元件结合，无法激活下游基因表达；当有四环素存在时，rtTA与TRE在四环素的作用结合，激活下游基因的表达。
dCas9(nuclease deactivated Cas9)是Cas9的突变体形式，可以在gRNA介导下识别靶标序列，但dCas9没有对靶标序列的切割活性，只有特异识别结合DNA的活性。dCas9可以与多种激活元件融合表达时，可以有效激活靶标基因的表达。
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以及dCas9的靶标序列至少之一。5×UAS是5个拷贝的Gal4VP16调控系统的响应元件。UAS序列中与Gal4蛋白结合的核心序列是一段4个拷贝串联的重复序列，每个拷贝包含17个碱基对，序列为5‘-CGGAGTACTGTCGTGGG-3’。7×tetO是7个拷贝的TRE是四环素调控系统的响应元件的识别序列。四环素响应元件(Tet Response Element TRE)包含7个重复的19个核苷酸的四环素操纵子序列5’-TCCCTATCAGTGATAGAGA-3’，可以被四环素诱导蛋白识别。
根据本发明的实施例，所述第一启动子与所述第二启动子为miniCMV。 5×UAS-miniCMV是一种合成的诱导型启动子。5×UAS是Gal4蛋白的识别序列，miniCMV是巨细胞病毒(Cytomegalovirus)启动子的部分序列。TRE也是一种诱导型启动子。7×tetO-miniCMV,tetO是四环素系统的响应元件的识别序列。
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LacI，tetR,zinc finger(锌指),KRAB、dCas9-KRAB至少之一。乳糖抑制子(Lactose repressor，LacI)通过自身DNA结合结构域的螺旋-转角-螺旋元件识别特异序列的方式与乳糖操纵子(Lactoseoperon LacO)的大沟结合，同时通过对称性相关的α-螺旋残基与LacO小沟的碱基序列紧密相连。这种紧密结合使RNA聚合酶高亲和性的结合在启动子区无法进入延伸状态，从而阻止mRNA转录和下游基因的表达。四环素抑制子(tetracycline repressor tetR)TetR含有保守的螺旋-转角-螺旋DNA结合区域(形成同型二聚体)，结合在四环素操纵子(tetracycline operon tetO)上，抑制下游基因的转录表达。Zinc finger锌指蛋白，由一个含有大约30个氨基酸的环和一个与环上的4个Cys或2个Cys和2个His配位的Zn2+构成，形成的结构像手指状。锌指蛋白是一类，对基因调控起重要的作用。根据其保守结构域的不同，可将锌指蛋白主要分为C2H2型、C4型和C6型。锌指通过与靶分子DNA、RNA、DNA－RNA的序列特异性结合，以及与自身或其他锌指蛋白的结合，在转录和翻译水平上调控基因的表达、细胞分化以及胚胎发育KRAB区域(The Krüppel associated box(KRAB)domain)是一类在人类近400锌指蛋白中存在的转录抑制区域。KRAB一般由75个氨基酸残基组成，最小的抑制有效区由45个氨基酸组成。它通过蛋白间以两亲性螺旋连接的方式发挥功能。可以被分成A、B两个区域,它们由不同的外显子编码而来,其中A box在转录抑制中起着核心作用,B box对A box的转录抑制功能起加强作用。
dCas9(nuclease deactivated Cas9)是Cas9的突变体形式，可以在gRNA介导下识别靶标序列，但dCas9没有对靶标序列的切割活性，只有特异识别结合DNA的活性。当dCas9与抑制型区域如何表达时可以有效地抑制靶标基因的表达。
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)、dCas9的靶标序列至少之一。tetO(tetracycline operon tetO)四环素操纵子，可以被四环素抑制子识别抑制下游基因的表达。LacO(Lactose operon LacO)乳糖操纵子，可以被乳糖抑制子(Lactose repressor LacI)识别抑制下游基因的表达。锌指蛋白目的序列(zinc finger target site)可以被锌指蛋白识别调控下游基因的表达。dCas9靶标序列可以被dCas9-KRAB在互补gRNA的协助下识别并抑制下游基因的表达。
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，乳糖抑制子(Lactose repressor LacI)通过自身DNA结合结构域的螺旋-转角-螺旋元件识别特异序列的方式与乳糖操纵子(Lactoseoperon LacO)的大沟结合，同时通过对称性相关的α-螺旋残基与LacO小沟的碱基序列紧密相连。这种紧密结合使RNA聚合酶高亲和性的结合在启动子区无法进入延伸状态，从而阻止mRNA转录和下游基因的表达。根据本发明的实施例，发明人将LacO构建在5×UAS-miniCMV启动子的miniCMV序列的两侧，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。但在此位置发明人并不排除使用其它抑制系统。
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。本发明的实施案例选用的抑制系统为tetR-KRAB/tetO系统，四环素抑制子(tetracycline repressor tetR)TetR含有保守的螺旋-转角-螺旋DNA结合区域(形成同型二聚体)，结合在四环素操纵子(tetracycline operon tetO)上，抑制下游基因的转录表达。在本实施案例中，发明人将tetO构建在5×UAS-miniCMV启动子的miniCMV序列的两侧，通过实验表明该系统可以有效的抑制启动子下游基因的表达。第一调控蛋白和第二调控蛋白组成的开关系统可以有效地相应输入信号，有效的区分不同细胞系。但在此处发明人并不排除使用其他抑制系统。
根据本发明的实施例，所述第五核酸分子和第九核酸分子的至少之一进一步包括编码目的蛋白的序列。利用根据本申请实施例的表达系统，可实现目的蛋白在特异细胞微环境中的特异性表达，实现了对目的蛋白表达的双向调控。根据本发明的具体实施例，本申请所述的表达系统对目的蛋白的表达调控的特异性和力度均显著提高。
根据本发明的实施例，所述第五核酸分子包括编码所述目的蛋白的序列，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。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，进而促进对肿瘤细胞等特异性细胞的免疫杀伤。
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。按转录时间的先后，腺病毒基因主要分为早期表达基因(E1～4)和晚期表达基因(L1～5)。腺病毒基因组进入细胞核后，细胞转录因子首先与E1A区上游的增强子结合，表达E1A蛋白，该蛋白的作用是调节细胞代谢，使病毒DNA更易于在细胞中复制。E1A蛋白还可以激活其他早期基因(E1B、E2A、E2B、E3和E4)的启动子，其中E2B驱动另外三个与病毒复制有关的早期基因转录单位末端蛋白前体(pTP,precursor terminal protein)、单链DNA结合蛋白(ssDBP,single-stranded DNA binding proteins) 以及DNA聚合酶(DNA pol,DNA polymerase)的表达，这三个基因的表达产物紧密地结合成一个复合物，与至少三种细胞蛋白相互作用，启动病毒基因组的复制。E1区基因表达产物分为E1A和E1B。E1A主要由两种成分构成，289R(或13S)和243R(或12S)。E1A蛋白的主要功能是调节细胞代谢，使细胞对病毒复制更易感。E1B19K与细胞Bcl-2基因的表达产物同源，可以通过灭活和清除Bax家族成员来防止细胞发生凋亡或坏死。E1B55K基因产物可以下调p53基因的转录水平，其他腺病毒基因(如E4或f6)也参与了这一过程。E1B55K基因产物还与病毒复制、病毒晚期mRNA的转录以及病毒RNA的转运有关。E2区基因表达产物分为E2A和E2B。其中，E2A即DNA结合蛋白(DBP，DNA Binding Protein)；E2B主要产物有两种，分别是末端蛋白前体(pTP)和病毒DNA聚合酶(pol)。三种蛋白与至少三种细胞内的因子相互作用，启动腺病毒DNA复制以及病毒晚期基因的转录和翻译过程。E4区的基因产物通常被称为orf 1～6/7，主要与病毒信使RNA的代谢有关。还有促进病毒DNA复制以及关闭宿主蛋白合成的功能。研究发现，一些E4产物可以与DNA激活的蛋白激酶结合，防止病毒DNA发生串联。由于该激酶可以激活p53基因，因此可以认为一些E4区基因产物可以抑制细胞凋亡。许多E1B和E4基因产物都与拮抗E1A蛋白功能有关。比如，E4抑制E1A对E2F启动子的激活。
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至少之一或者这些因子的融合表达形式。其中，PD-1(programmed death 1)程序性死亡受体1，是一种重要的免疫抑制分子。为CD28超家族成员,其最初是从凋亡的小鼠T细胞杂交瘤2B4.11克隆出来。PD-1是T细胞表面的重要抑制分子，其胞内段含有一个免疫受体酪氨酸抑制基序ITIM和一个免疫受体酪氨酸转换基序(ITSM)。ITSM介导了蛋白酪氨酸磷酸酶家族磷酸酶的募集以及对T细胞活化信号的抑制，其配体为PD-L1和PD-L2主要在免疫系统效应期的肿瘤微环境中发挥抑制T细胞活化的重要作用。细胞程式死亡-配体1(Programmed cell death 1ligand 1，PD-L1)也称为表面抗原分化簇274(cluster of differentiation 274，CD274)或B7同源体(B7homolog 1，B7-H1)，是人类体内的一种蛋白质，由CD274基因编码。PD-L1在多种肿瘤细胞和肿瘤细胞微环境中造血细胞中有诱导性表达。其表达水平与某些肿瘤的恶性程度呈正相关。PD-1抗体与PD-L1抗体可以阻断PD-1与PD-L1的结合，组织肿瘤细胞通过PD-1/PD-L1通路进行免疫逃逸。CTLA-4的全称叫做细胞毒性T细胞相关蛋白-4(Cytotoxic T lymphocyte associate protein-4)，它是T细胞表面表达的一类共刺激分子(co-stimulatory molecule)。与CD28的功能类似，在T细胞激活过程中，CTLA-4能够与APC表面的CD80/CD86特异性结合来激活下游信号。研究发现，T细胞中主要表达CTLA-4的细胞为调节性T细胞(Treg)，一类可以负向调节细胞免疫的T细胞；在缺 失CTLA-4受体时，小鼠表现出T细胞过度活化伴随严重的自身免疫病。以上结果可以得出，Treg需要通过CTLA-4行使其功能。另外，CTLA-4也在conT细胞中有表达，其作用是抑制T细胞激活的信号传递。T细胞免疫球蛋白以及黏蛋白结构与分子(T cell immunoglobulin and mucin-domain containing molecule,TIM)基因家族是Mclntire在2001年寻找小鼠哮喘病易感基因的研究时发现并通过基因组分析及克隆定位鉴定的新的基因家族，该家族基因结构含有免疫球蛋白V区和黏蛋白区。Tim-3是TIM家族中的重要成员，表达在活化的Th1细胞表面的负向调节分子。目前在CD8+T细胞、Th17细胞、Treg、NK细胞和其他淋巴细胞亚群也发现了TIM3的表达。GM-CSF(Granulocyte-macrophage Colony Stimulating Factor)粒细胞-巨噬细胞集落刺激因子是一种由巨噬细胞，T细胞，NK细胞等分泌的单体糖蛋白细胞因子，GM-CSF刺激干细胞产生粒细胞(中性粒细胞、嗜酸性粒细胞和嗜碱性粒细胞和单核细胞)。GM-CSF也会影响免疫系统中的成熟细胞如抑制中性粒细胞的转移，改变细胞表面的受体表达。该因子还可以通过激活巨噬细胞抑制真菌的感染。IL-2(interleukin 2)白细胞介素2能介导白细胞与白细胞之间以及白细胞与其他细胞之间相互作用的细胞因子。IL-2主要由活化的T细胞产生，以自分泌与旁分泌的方式作用于局部的靶细胞，是参与免疫应答的主要细胞因子，具有明显的免疫效应。可促进T细胞增殖以及产生细胞因子，促进B细胞增值和分泌Ig,激活巨噬细胞，增强NK细胞活化和增殖等。此外，IL-2亦具有负向调节作用，可诱导Ag活化的T细胞发生凋亡，限制免疫应答的强度，避免发生明显的免疫损伤。白细胞介素IL-12(interlukin-12)是一种由树突状细胞，巨噬细胞、中性粒细胞、人B淋巴母细胞(nc-37)在响应抗原刺激的条件下产生的白细胞介素。IL-12被称为T细胞刺激因子，参与了原初细胞向Th1的分化过程，刺激干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)的产生。IL-12在调节自然杀伤细胞和T淋巴细胞的活性中起重要作用。IL-12介导NK细胞和CD8+细胞毒性T淋巴细胞的细胞毒活性增强。IL-12也具有抗血管生成活性，这意味着它可以阻断新生血管的形成。它通过增加γ-干扰素的生产增加诱导蛋白10(IP-10或CXCL10)。IP-10然后介导的抗血管生成作用。由于其能够诱导免疫应答和抗血管生成活性，人们一直在测试IL-12作为一种可能的抗癌药物的。IL-15是新近发现的一种因子，可由活化的单核－巨噬细胞、表皮细胞和成纤维细胞等多种细胞产生。IL-15的分子结构与IL-2有许多相似之外，因此可以利用IL-2受体的β链和γ链与靶细胞结合，发挥类似IL-2的生物学活性。IL-15可诱导B细胞增殖和分化，是唯一能部分取代IL-2诱导初期抗体产生的细胞因子；IL-15能够刺激T细胞和NK细胞增殖，诱导LAK细胞活性，还能与IL-12协同刺激NK细胞产生IFN-γ。
根据本发明的实施例，所述目标蛋白与所述第一调控蛋白是在同一启动子的调控下共表达的，并且所述目标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。目标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，目标蛋白与 第一调控蛋白分开，目标蛋白和第一调控蛋白相互独立地发挥功能。
根据本发明的实施例，所述第九核酸分子与所述第十核酸分子分别独立地通过下列方式抑制所述第一调控蛋白或所述第二调控蛋白的表达：第九与第十核酸分子通过RNA干扰的方式抑制第一和第二调控蛋白的表达，其中，microRNA为不同细胞微环境表达的特异性microRNA，第九或第十核酸分子为microRNA的特异性靶标序列，进而通过RNA干扰的方式,可实现在特异性微环境中表达的microRNA对靶标序列的特异性作用，进而实现对第一调控蛋白或第二调控蛋白表达的特异性调控。MicroRNA(miRNA)是一类内生的、长度约20-24个核苷酸的小RNA，几个miRNAs也可以调节同一个基因。可以通过几个miRNAs的组合来精细调控某个基因的表达。MicroRNA存在多种形式，最原始的是pri-miRNA，长度大约为300～1000个碱基；pri-miRNA经过一次加工后，成为pre-miRNA即microRNA前体，长度大约为70～90个碱基；pre-miRNA再经过Dicer酶酶切后，成为长约20～24nt的成熟miRNA。microRNA介导(RNA-induced silencing complex RISC)抑制靶标基因的表达。microRNA-RISC对靶基因mRNA的作用一直主要取决于它与靶基因转录体序列互补的程度，有三种方式。第一种是切断靶基因的mRNA分子——miRNA与靶基因完全互补结合，作用方式和功能与siRNA非常相似，最后切割靶mRNA。在植物中，大部分miRNA都以这种方式，靶基因mRNA断裂后，无poly(A)的分子的3‘端加上多个U并很快降解，含poly(A)的分子能稳定存在一段时间(如拟南芥miR-171)。在植物中目前有一个miRNA和3个潜在的目标靶基因完全互补，尽管还不清楚这些基因是否就是miRNA的目标靶，这也是第一次发现miRNA和其潜在的目标靶完全互补，也提示miRNA可能包含和siRNA类似的作用方式。第二种是抑制靶基因的翻译——作用时与靶基因不完全互补结合，进而阻遏翻译而不影响mRNA的稳定性，这种miRNA是目前发现最多的种类(如线虫lin-4)。而在植物中极少数的miRNA通过此方式来抑制靶基因。第三种是结合抑制——具有以上两种作用模式：当与靶基因互补结合时，直接靶向切割mRNA；当与靶基因不完全结合时，起调节基因表达的作用。
根据本发明的实施例，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特异性识别第二microRNA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。进而，第一调控蛋白在异常细胞中表达，在正常细胞中不表达或低表达，而第二调控蛋白在正常细胞中表达，在异常细胞中不表达或低表达。
根据本发明再一具体的实施例，所述第一microRNA包括选自下列的至少之一：miR199a、miR95、miR125、miRamir25b、Let-7、miR143、miR145以及miR200C。上述microRNA在正常肝细胞中表达。
根据本发明的再一具体示例，所述第二microRNA包括选自下列的至少之一：肝癌细胞(HepG2,Huh7,PLC)中表达的miR21、miR223、miR224、miR221、miR18、miR214、miR146a以及miR1792。上述第二microRNA为肝癌细胞特异性表达的microRNA。进而，第一调控蛋白在肝癌细胞中表达，在正常细胞中不表达或低表达，而第二调控蛋白在正常细胞中表达，在肝癌细胞中不表达或低表达。
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可选的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可选的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，在适宜的微环境，如细胞中，实现对目的基因的特异性表达的调控。
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适宜微环境中的功能的发挥即可。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：
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。脂质体载体是用人造双层磷脂质，包装DNA后形成的脂质体-DNA复合物。脂质体载体无毒无抗原性，目的基因与脂质体的包裹使其可以免受核酸酶降解，容量大，可单独或联合其他载体使用，并可通过静脉注射使目的基因进入特定部位。缺点：表达时间短，不能通过细胞膜屏障。分子耦联载体：分子耦联载体由DNA、DNA结合因子和配体三部分组成。
根据本发明的实施例，所述病毒包括选自腺病毒、牛痘病毒、逆转录病毒的至少之一。
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是同一个载体。根据本发明的具体实施例，发明人通过Cascade Golden-Gate Gibson/Gateway Assemble Method技术将本申请表达系统中的元件负载在同一表达载体上，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。
根据本发明的实施例，所述同一个载体为腺病毒。腺病毒作为基因治疗载体具有如下优点：1)宿主范围广,对人致病性低。腺病毒载体系统可广泛用于人类及非人类蛋白的表达。腺病毒可感染一系列哺乳动物细胞，因此在大多哺乳动物细胞和组织中均可用来表达重组蛋白。特别需要指出的是：腺病毒具有嗜上皮细胞性，而人类的大多数的肿瘤就是上皮细胞来源的。另外，腺病毒的复制基因和致病基因均已相当清楚，在人群中早已流行(70-80％成人体内都有腺病毒的中和抗体存在)。人类感染野生型腺病毒后仅产生轻微的自限性症状，且病毒唑治疗有效。2)在增殖和非增殖细胞中感染和表达基因。逆转录病毒只能感染增殖性细胞，因此DNA转染不能在非增殖细胞中进行，而必须使细胞处于持续培养状态。 腺病毒则能感染几乎所有的细胞类型，除了一些抗腺病毒感染的淋巴瘤细胞。腺病毒是研究原代非增殖细胞基因表达的最佳系统，它可以使转化细胞和原代细胞中得到的结果直接进行对比。3)能有效进行增殖，滴度高。腺病毒系统可产生1010到1011VP/mlL，浓缩后可达1013VP/mL，这一特点使它非常适用于基因治疗。4)与人类基因同源。腺病毒载体系统一般应用人类病毒作为载体，以人类细胞作为宿主，因此为人类蛋白进行准确的翻译后加工和适当的折叠提供了一个理想的环境。大多数人类蛋白都可达到高水平表达并且具有完全的功能。5)不整合到染色体中，无插入致突变性。逆转录病毒可随机整合到宿主染色体，导致基因失活或激活癌基因。而腺病毒则除了卵细胞以外几乎在所有已知细胞中都不整合到染色体中，因此不会干扰其它的宿主基因。在卵细胞中整合单拷贝病毒则是产生具有特定特征的转基因动物的一个较好的系统。6)能在悬浮培养液中扩增。293细胞可以适应悬浮培养，这一调整可使病毒大量扩增。大量事实证明悬浮293细胞可在1～20L的生物反应器中表达重组蛋白。7)能同时表达多个基因。这是第一个可以在同一细胞株或组织中用来设计表达多个基因的表达系统。最简单的方法是将含有两个基因的双表达盒插入腺病毒转移载体中，或者用不同的重组病毒共转染目的细胞株来分别表达一个蛋白。测定不同重组病毒的MOI比值可正确估计各重组蛋白的相对共表达情况。
根据本发明的具体实施例，发明人将上述腺病毒注入肿瘤小鼠模型，可显著抑制小鼠肿瘤的生长。上述腺病毒可作为一种安全有效的溶瘤病毒疫苗，实现对相关肿瘤的安全有效地特异性杀伤。
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。具体地，发明人通过三步将识别癌症细胞的基因线路插入到腺病毒载体上。首先将区分正常细胞与癌症细胞的相关标志元件如肿瘤特异性启动子，基因线路构建相关的抑制元件，区分癌症细胞的微小RNA的识别序列构建到一级载体质粒上，构成一级元件库。元件两端设计有Esp3I的识别位点，用于通过Goldeng Gate的方式将多个一级元件构建到二级表达系统上。第二步，构建表达系统库，从一级库中选取多个一级元件通过Golden Gate的方式将他们组装成三个表达系统，包括肿瘤特异性启动子系统，第一调控元件所介导的抑制系统和第二调控元件所介导的抑制系统。每个表达系统的两端设计有BsaI内切酶的识别位点。第三步，组装基因线路，通过Golden Gate的方式随机的将三种表达系统组装起来形成完整的基因线路。基因线路两端设计有用于下一步实验的Gibson同源序列或者Gateway识别位点。最后通过Gibson或者Gateway的方式将基因线路构建到已经去除了E1和部分E3的腺病毒载体上。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。
重组病毒
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，所述肿瘤细胞特异性启动子为甲胎蛋白特异性启动子；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，所述第一识别序列为5×UAS；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与病毒复制蛋白共表达的，并且所述效应因子之间通过可切割的连接肽连接，所述目标蛋白与所述第一调控蛋白由同一启动子调控共表达的，并且所述目标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，所述第二识别序列为5×UAS；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。利用根据本申请实施例的重组病毒，在甲胎蛋白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、目标蛋白在肿瘤细胞中特异性表达，第二调控蛋白tetR-KRAB在肿瘤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、目标蛋白在第一启动子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，可实现蛋白在肿瘤细胞中更加特异性的表达(如目标蛋白E1A，第一调控蛋白LacI)或不表达(如第二调控蛋白tetR-KRAB)，在肿瘤细胞中的表达或不表达效率和特异性更高。
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。⑴逆转录病毒载体的特点：结构和感染过程与普通逆转录病毒相似；可使靶细胞变成稳定表达目的基因的转化细胞；感染靶细胞后不扩散；假病毒感染靶细胞的效率非常高；不感染非增殖细胞。⑵腺病毒载体的特点：宿主范围广；腺病毒蛋白表达不以宿主增殖为必要条件；可获得高病毒效价；重组体非常稳定；不会引起肿瘤；有较高的安全性；无包膜，不易被补体灭活，可直接在体内应用；不整合入染色体。⑶单纯疱疹病毒载体的特点：滴度高；容量大；增殖细胞和非增殖细胞均可感染；不整合，但可长期存在并可稳定表达。
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很多优点：1.宿主范围广,对人致病性低。腺病毒载体系统可广泛用于人类及非人类蛋白的表达。腺病毒可感染一系列哺乳动物细胞，因此在大多哺乳动物细胞和组织中均可用来表达重组蛋白。特别需要指出的是：腺病毒具有嗜上皮细胞性，而人类的大多数的肿瘤就是上皮细胞来源的。另外，腺病毒的复制基因和致病基因均已相当清楚，在人群中早已流行(70-80％成人体内都有腺病毒的中和抗体存在)。人类感染野生型腺病毒后仅产生轻微的自限性症状，且病毒唑治疗有效。2.在增殖和非增殖细胞中感染和表达基因。逆转录病毒只能感染增殖性细胞，因此DNA转染不能在非增殖细胞中进行，而必须使细胞处于持续培养状态。腺病毒则能感染几乎所有的细胞类型，除了一些抗腺病毒感染的淋巴瘤细胞。腺病毒是研究原代非增殖细胞基因表达的最佳系统，它可以使转化细胞和原代细胞中得到的结果直接进行对比。3.能有效进行增殖，滴度高。腺病毒系统可产生1010到1011VP/mL，浓缩后可达1013VP/mlL，这一特点使它非常适用于基因治疗。4.与人类基因同源。腺病毒载体系统一般应用人类病毒作为载体，以人类细胞作为宿主，因此为人类蛋白进行准确的翻译后加工和适当的折叠提供了一个理想的环境。大多数人类蛋白都可达到高水平表达并且具有完全的功能。5.不整合到染色体中，无插入致突变性。逆转录病毒可随机整合到宿主染色体，导致基因失活或激活癌基因。而腺病毒则除了卵细胞以外几乎在所有已知细胞中都不整合到染色体中，因此不会干扰其它的宿主基因。在卵细胞中整合单拷贝病毒则是产生具有特定特征的转基因动物的一个较好的系统。6.能在悬浮培养液中扩增。293细胞可以适应悬浮培养，这一调整可使病毒大量扩增。大量事实证明悬浮293细胞可在1～20L的生物反应器中表达重组蛋白。7.能同时表达多个基因。这是第一个可以在同一细胞株或组织 中用来设计表达多个基因的表达系统。最简单的方法是将含有两个基因的双表达盒插入腺病毒转移载体中，或者用不同的重组病毒共转染目的细胞株来分别表达一个蛋白。测定不同重组病毒的MOI比值可正确估计各重组蛋白的相对共表达情况。
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-12、IL-15、GM-CSF至少之一或者这些因子的融合表达形式。PD-1(programmed death 1)程序性死亡受体1，是一种重要的免疫抑制分子。为CD28超家族成员,其最初是从凋亡的小鼠T细胞杂交瘤2B4.11克隆出来。PD-1是T细胞表面的重要抑制分子，其胞内段含有一个免疫受体酪氨酸抑制基序ITIM和一个免疫受体酪氨酸转换基序(ITSM)。ITSM介导了蛋白酪氨酸磷酸酶家族磷酸酶的募集以及对T细胞活化信号的抑制，其配体为PD-L1和PD-L2主要在免疫系统效应期的肿瘤微环境中发挥抑制T细胞活化的重要作用。细胞程式死亡-配体1(Programmed cell death 1ligand 1，PD-L1)也称为表面抗原分化簇274(cluster of differentiation 274，CD274)或B7同源体(B7homolog 1，B7-H1)，是人类体内的一种蛋白质，由CD274基因编码。PD-L1在多种肿瘤细胞和肿瘤细胞微环境中造血细胞中有诱导性表达。其表达水平与某些肿瘤的恶性程度呈正相关。PD-1抗体与PD-l1抗体可以阻断PD-1与PD-L1的结合，组织肿瘤细胞通过PD-1/PD-L1通路进行免疫逃逸。CTLA-4的全称叫做细胞毒性T细胞相关蛋白-4(Cytotoxic T lymphocyte associate protein-4)，它是T细胞表面表达的一类共刺激分子(co-stimulatory molecule)。与CD28的功能类似，在T细胞激活过程中，CTLA-4能够与APC表面的CD80/CD86特异性结合来激活下游信号。研究发现，T细胞中主要表达CTLA-4的细胞为调节性T细胞(Treg)，一类可以负向调节细胞免疫的T细胞；在缺失CTLA-4受体时，小鼠表现出T细胞过度活化伴随严重的自身免疫病。以上结果可以得出，Treg需要通过CTLA-4行使其功能。另外，CTLA-4也在conT细胞中有表达，其作用是抑制T细胞激活的信号传递。T细胞免疫球蛋白以及黏蛋白结构与分子(T cell immunoglobulin and mucin-domain containing molecule,TIM)基因家族是Mclntire在2001年寻找小鼠哮喘病易感基因的研究时发现并通过基因组分析及克隆定位鉴定的新的基因家族，该家族基因结构含有免疫球蛋白V区和黏蛋白区。TIM3是TIM家族中的重要成员，表达在活化的Th1细胞表面的负向调节分子。目前在CD8+T细胞、Th17细胞、Treg、NK细胞和其他淋巴细胞亚群也发现了TIM3的表达。GM-CSF(Granulocyte-macrophage Colony Stimulating Factor)粒细胞-巨噬细胞集落刺激因子是一种由巨噬细胞，T细胞，NK细胞等分泌的单体糖蛋白细胞因子，GM-CSF刺激干细胞产生粒细胞(中性粒细胞、嗜酸性粒细胞和嗜碱性粒细胞和单核细胞)。GM-CSF也会影响免疫系统中的成熟细胞如抑制中性粒细胞的转移，改变细胞表面的受体表达。该因子还可以通过激活巨噬细胞抑制真菌的感染。 IL-2(interleukin 2)白细胞介素2能介导白细胞与白细胞之间以及白细胞与其他细胞之间相互作用的细胞因子。IL-2主要由活化的T细胞产生，以自分泌与旁分泌的方式作用于局部的靶细胞，是参与免疫应答的主要细胞因子，具有明显的免疫效应。可促进T细胞增殖以及产生细胞因子，促进B细胞增值和分泌Ig,激活巨噬细胞，增强NK细胞活化和增殖等。此外，IL-2亦具有负向调节作用，可诱导Ag活化的T细胞发生凋亡，限制免疫应答的强度，避免发生明显的免疫损伤。白细胞介素IL-12(interlukin-12)是一种由树突状细胞，巨噬细胞、中性粒细胞、人B淋巴母细胞(nc-37)在响应抗原刺激的条件下产生的白细胞介素。IL-12被称为T细胞刺激因子，参与了原初细胞向Th1的分化过程，刺激干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)的产生。IL-12在调节自然杀伤细胞和T淋巴细胞的活性中起重要作用。IL-12介导NK细胞和CD8+细胞毒性T淋巴细胞的细胞毒活性增强。IL-12也具有抗血管生成活性，这意味着它可以阻断新生血管的形成。它通过增加γ-干扰素的生产增加诱导蛋白10(IP-10或CXCL10)。IP-10然后介导的抗血管生成作用。由于其能够诱导免疫应答和抗血管生成活性，人们一直在测试IL-12作为一种可能的抗癌药物的。IL-15是新近发现的一种因子，可由活化的单核－巨噬细胞、表皮细胞和成纤维细胞等多种细胞产生。IL-15的分子结构与IL-2有许多相似之外，因此可以利用IL-2受体的β链和γ链与靶细胞结合，发挥类似IL-2的生物学活性。IL-15可诱导B细胞增殖和分化，是唯一能部分取代IL-2诱导初期抗体产生的细胞因子；IL-15能够刺激T细胞和NK细胞增殖，诱导LAK细胞活性，还能与IL-12协同刺激NK细胞产生IFN-γ。
重组细胞
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，攻击异种细胞，如肿瘤细胞，安全性高，特异性强。
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。
在制备药物中的用途
在本发明的第四方面，本方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，所述药物用于治疗癌症。本申请所述的表达系统能够实现目的蛋白在肿瘤细胞中特异性表达，根据本申请实施例的药物对癌症的治疗效果更加有效、特异性更强、安全性更高。在本实施案例中，发明人用合成生物学的理念设计响应多靶点的基因线路调控腺病毒转录相关的关键基因E1A的表达。同时共表达可以激活系统免疫反应的相关细胞因子或抗体基因。首先，与传统的溶瘤腺病毒不同，发明人设计的基因线路可以在多层次调控腺病毒的包装。第一步，发明人利用肿瘤特异性的启动子调 控基因线路的总开关Gal4VP16的表达，第二步，基因线路内部microRNA靶标序列可以响应不同细胞系中microRNA的表达来区分肿瘤细胞和非肿瘤细胞。第三步，发明人设计的基因线路采用的双抑制系统十分稳定，可以高效的响应外部输入信号如细胞特异性启动子和微小RNA信号，并且该基因线路可以进一步扩大输入信号的差异，更加高效的区分肿瘤细胞和非肿瘤细胞。第四步，在发明人的腺病毒载体中本系统去除了腺病毒的E1B基因，E1B基因可以与正常细胞的P53基因相互作用保证腺病毒在细胞中的顺利增殖。去除E1B基因后，腺病毒无法在表达P53的正常细胞中增殖。然而，在很多肿瘤细胞中缺失P53基因的表达，不表达E1B的腺病毒仍然可以有效的扩增。所以通过多角度多水平的调控，本实施例中的腺病毒载体相较于传统的溶瘤腺病毒特异性更强，安全性也更好。另外，本实施例还利用这一腺病毒作为载体携带表达多种细胞因子和抗体基因。在腺病毒的溶瘤的同时利用这些共表达的因子激活机体系统性的免疫反应，进一步提高溶瘤腺病毒的效果。
具体地，本专利利用合成生物学手段构建基因线路调控溶瘤腺病毒在肝细胞癌中的特异表达包装。除了利用腺病毒的溶瘤作用，发明人设计构建的腺病毒还装载了可以激活机体免疫反应的细胞因子，通过表达这些细胞因子，激发机体产生抑制肿瘤生长的免疫反应达到治疗肿瘤的目的。本专利设计的系统具有很大的创新性和技术优势：
高效安全：本专利设计的基因线路综合利用多层次多水平的区分正常细胞与癌细胞的生物标志物，同时利用双抑制的闭合回路基因线路调控腺病毒在不同细胞中的复制。通过这个基因线路可以更加敏感的响应不同细胞系微环境，同时减少基因表达噪音影响。
高效：本专利利用本基因线路搭载多种细胞因子和/或抗体序列，从而达到在目的细胞中引起有效的免疫反应。
模块化组建基因线路：首先将基因线路中涉及的生物标志物模块化，将每一模块建库，库中包含针对不同疾病的生物标志物。通过快速组建相关模块，搭建针对特异病症的基因线路。
精准：快速拼装的技术为发明人替换不同肿瘤标志物提供了可能，可以针对不同病人进行不同的基因线路设计，实现精准治疗
根据本发明的实施例，所述癌症包括肝癌、肺癌、结直肠癌，黑色素瘤、乳腺癌或前列腺癌。发明人发现，根据本发明实施例的药物对肝癌、肺癌、结直肠癌，黑色素瘤、乳腺癌或前列腺癌的治疗效果更加显著。
根据本发明的具体实施例，发明人发现microRNA在不同肿瘤细胞系中的表达情况如表1所示。向上的箭头表明与正常细胞相比在癌症细胞中高表达的microRNA，向下的箭头表明与正常细胞相比在癌症细胞中低表达的microRNA。
表1：
肝细胞癌 miR-21↑，18↑，224↑，199↓，195↓，200↓，125↓
肺癌 let-7↓，miR-17-92↑
乳腺癌 miR125b↓，145↓，21↓，155↓
颅脑肿瘤 miR-21↑，221↑，181↓，
结直肠癌 miR143↓，145↓
淋巴癌 miR155↑，miR-17-92↑
黑色素瘤 miR21↑，221↑，214↑146a↑137↓200c↓let-7↓
利用表达系统表达目的蛋白的方法
在本发明的第五方面，本发明提出了一种利用表达系统表达目的蛋白的方法，所述表达系统为前面所述的表达系统。根据本发明的实施例，所述方法包括：(1)使所述第五核酸分子包含编码所述目的蛋白的核酸序列；(2)使所述第十核酸分子抑制所述第二调控蛋白的表达，以便表达所述目的蛋白。利用根据本发明实施例的上述表达目的蛋白的方法，第二调控蛋白介导的对第一启动子的抑制机制解除，目的蛋白在细胞特异性启动子和第一启动子的共同作用下，在特异细胞中高效表达。
根据本发明的实施例，所述表达是在细胞中进行的。细胞可以提供目的蛋白表达的微环境，目的蛋白在细胞中的表达更加高效。
根据本发明的实施例，所述第十核酸分子含有被第二microRNA特异性是别的核酸序列，在步骤(2)中包括使所述第二microRNA与所述第十核酸分子接触。MicroRNA(miRNA)是一类内生的、长度约20-24个核苷酸的小RNA。多个miRNAs也可以调节同一个基因，可以通过几个miRNAs的组合来精细调控单个基因的表达。MicroRNA存在多种形式，最原始的是pri-miRNA，长度大约为300～1000个碱基；pri-miRNA经过一次加工后，成为pre-miRNA即microRNA前体，长度大约为70～90个碱基；pre-miRNA再经过Dicer酶酶切后，成为长约20～24nt的成熟miRNA。microRNA介导(RNA-induced silencing complexRISC)抑制靶标基因的表达。microRNA-RISC对靶基因mRNA的作用一直主要取决于它与靶基因转录体序列互补的程度，有三种方式。第一种是切断靶基因的mRNA分子——miRNA与靶基因完全互补结合，作用方式和功能与siRNA非常相似，最后切割靶mRNA。在植物中，大部分miRNA都以这种方式，靶基因mRNA断裂后，无poly(A)的分子的3‘端加上多个U并很快降解，含poly(A)的分子能稳定存在一段时间(如拟南芥miR-171)。在植物中目前有一个miRNA和3个潜在的目标靶基因完全互补，尽管还不清楚这些基因是否就是miRNA的目标靶，这仍是第一次发现miRNA和其潜在的目标靶完全互补，也提示miRNA可能包含和siRNA类似的作用方式。第二种是抑制靶基因的翻译——作用时与靶基因不完全互补结合，进而阻遏翻译而不影响mRNA的稳定性，这种miRNA是目前发现最多的种类(如线虫lin-4)。而在植物中极少数的miRNA通过此方式来抑制靶基因。第三种是结合抑制——具有以上两种作用模式：当与靶基因互补结合时，直接靶向切割mRNA；当与靶基因不完 全结合时，起调节基因表达的作用。
在本发明的第六方面，本发明又提出了一种利用表达系统表达目的蛋白的方法，所述表达系统为前面所述的表达系统。根据本发明的实施例，所述方法包括：(1)使所述第八核酸分子包含编码第一目的蛋白的核酸序列，使所述第五核酸分子包含编码第二目的蛋白的核酸序列；(2)通过下列之一表达所述第一目的蛋白或所述第二目的蛋白：使所述第九核酸分子抑制所述第一调控蛋白的表达，以便表达所述第一目的蛋白；使所述第十核酸分子抑制所述第二调控蛋白的表达，以便表达所述第二目的蛋白。利用根据本发明实施例的上述表达目的蛋白的方法，第二调控蛋白介导的对第一启动子的抑制机制解除，第二目的蛋白在细胞特异性启动子和第一启动子的共同作用下，在特异细胞中高效表达或第一调控蛋白介导的对第二启动子的抑制机制解除，第一目的蛋白在细胞特异性启动子和第二启动子的共同作用下，在特异细胞中高效表达。
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，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。
实验材料与方法
1、细胞培养与细胞转染
HEK293(293-H)细胞系采购自Invitrogen公司，人的肝癌细胞系HepG2，正常肝细胞Chang购买于ATCC，人肝癌细胞Huh7购买于北纳创联，Hepa1-6购买于ATCC。细胞培养在高糖DMEM完全培养基内(Dulbecco's Modified Eagle's Medium)，4.5g/L葡萄糖，0.045unit/mL青霉素和0.045g/mL链霉素以及10％FBS(购自Invitrogen公司)，温度37℃，湿度100％，CO2浓度5％。
在细胞系转染实验中，取24孔板(Falcon公司)，向每孔中加入0.5mL含约7.5×104个HEK293细胞的高糖DMEM培养基培养24小时。在转染前，换液，加入新的DMEM完全培养基。使用Attractene转染试剂(Qiagen公司)或使用Lipofectamine LTX(Life technologies公司)进行转染实验。当使用Attractene时，将DNA按量混合后加入1.5μL Attractene，室温静置15分钟，之后加入到细胞内。当使用Lipofectamine LTX时，首先将DNA按量混合后加入适量Plus reagent后室温静置5分钟，之后加入1.25μL Lipofectamine LTX，室温静置25分钟，之后加入到细胞内。pDT7004(pUBI-linker-NOS)包含一个玉米泛素启动子(UBI)，下游为NOS终止子，在UBI与NOS之间没有任何编码蛋白序列，本申请用pDT7004配平不同实验组的质粒DNA。转染后培养48小时进行流式细胞术分析或 进行细胞因子检测相关实验。
2、腺病毒载体
本说明中实施案例使用了两种原始腺病毒载体序列：ViraPowerTM Adenoviral GatewayTM Expression Kit(K49300Invitrogen)；Adeno-X Adenoviral System 3(632266Clontech)。载体结构如图1和图2所示。
3、质粒构建
AFP启动子截短实验相关质粒构建；AFP enhancer序列与AFP启动子序列直接通过设计PCR引物从肝癌细胞系的基因组中PCR得到。AFP启动子序列中的点突变通过overlap PCR获得。AFP enhancer与AFP启动子序列构建在入门克隆序列中，两端设计有LR酶的识别序列，通过LR将特异性启动子与激活基因Gal4VP16构建在同一载体上。microRNA的靶向序列通过引物退火酶切连接的方式插入CMV-EYFP载体上(XbaI/SalI)。系统一级元件库是通过Golden Gate的方式构建的。IIs型限制性内切酶(如BsaI、BbsI、BsmBI和SapI等)是一类特殊的限制性内切酶，与分子生物学最常用的II型限制性内切酶(识别位点为回文对称序列，切割位点与识别序列重叠)不同，其识别位点是一个非回文对称的序列，而切割位点位于识别位点的外侧。因此，当被IIs型限制性内切酶酶切后的两个片段相连时，原来的识别位点将不复存在，在后续的酶切连接反应中也就不会再次被切割，也达到“焊死”的效果。Golden Gate克隆技术利用IIs型限制性内切酶的这一特性在这些酶的识别序列外侧人为地设计切割位点不同的序列，然后利用同一限制性内切酶产生不同的黏性末端，从而一次性组装多个片段，克服了传统多片段组装中需要同时使用多个不同限制性内切酶的不足。相对于传统酶切连接技术而言，该技术的最大特点是不会引入任何额外的“疤痕”，从而实现了DNA片段的无痕连接。本实施例中第一级元件库中使用的IIs型限制性内切酶为Esp3I。系统的二级逻辑线路库也是通过Golden Gate的方式构建的，使用的IIs型限制性内切酶为BsaI。逻辑线路通过Gateway方式插入到ViraPowerTM Adenoviral GatewayTM Expression Kit的腺病毒载体序列中；通过Gibson克隆方式插入到Adeno-X Adenoviral System 3的腺病毒载体序列中。Gateway技术是基于已研究的非常清楚的λ噬菌体位点特异重组系统(attB×attP→attL×attR)。BP和LR两个反应就构成了Gateway技术。BP反应是利用一个attB DNA片段或表达克隆和一个attP供体载体之间的重组反应，创建一个入门克隆。LR反应是一个attL入门克隆和一个attR目的载体之间的重组反应。LR反应用来在平行的反应中转移目的序列到一个或更多个目的载体。在本实施例中发明人采用了LR反应。Gibson assembly技术又被称为“Gibson恒温一步组装法”，是美国J.CraigVenter研究所的Gibson等创建的一种DNA组装方法，即利用T5核酸外切酶、DNA聚合酶及连接酶的协同作用在体外将多个带有末端重叠序列的DNA片段组装起来。其中，T5核酸外切酶 具有5’→3’核酸外切酶活性，能够从5’端切割有重叠区的DNA片段产生3’突出末端，然后该单链DNA的重叠序列在50℃特异性退火(此时T5核酸外切酶逐渐失活)，最后DNA聚合酶和Taq连接酶修复连接而成的双链DNA，从而形成完整的DNA分子，实现无缝拼接。
4、细胞RNA提取与反转录，定量PCR
利用Trizol法提取细胞RNA：先离心沉淀5×106个细胞，加1mL Trizol后，反复用枪吹打或剧烈振荡以裂解细胞。在室温15-30℃下放置5分钟，加0.2mL氯仿，用力震荡15秒，在室温下放置2～3分钟后，12000×g 4℃离心15分钟。取上层水相加0.5mL异丙醇，室温放置10分钟，12000×g 4℃离心10分钟。1mL 75％乙醇进行洗涤，涡旋混合，7500×g 4℃离心5分钟，弃上清；让沉淀的RNA在室温下自然干燥；用RNase-free水溶解RNA沉淀。反转录实验是利用QuantiTect Rev.Transcription Kit(Cat No./ID:205310 QIAGEN)进行。定量PCR引物有qAFP for：CAAAGCTGAAAATGCAGTTGAATG(SEQ ID NO：15)；qAFP rev：TTCCCCATCCTGCAGACAATCC(SEQ ID NO：16)；GAPDH for：AGAAGGCTGGGGCTCATTTG(SEQ ID NO：17)；GAPDH rev：AGGGGCCATCCACAGTCTTC(SEQ ID NO：18)。荧光染料为PowerUpTM
Figure PCTCN2017096043-appb-000040
Green Master Mix(A25741Thermo Fisher)。
5、腺病毒包装、纯化和滴度测定
提取高纯度无内毒素重组腺病毒质粒：PacI线性化后转染HEK293细胞，转染过夜更换为完全培养基，第一代培养一周左右，中间根据细胞生长状态添加培养基，然后收集细胞和1mL培养液置于15mL离心管后，液氮/37℃冻融三次，2000rpm离心5分钟，取上清即为病毒液初代原液。连续三代反复扩增收集病毒后进行病毒的大量扩增(10个15cm平板)，然后通过CsCl密度梯度离心-透析联用法纯化病毒。CsCl梯度的制备方法如下：加入2mL密度为1.4g/mL的CsCl溶液，然后缓慢加入3mL密度为1.3g/mL的CsCl溶液，再加入5mL的病毒悬浮液。20000rpm 4℃离心10小时。收集密度在1.3g/mL和1.4g/mL之间的病毒条带至透析袋中(透析袋使用前用10mM的EDTA-Na2煮沸10分钟)。在透析缓冲液(50g蔗糖，10mL 1M Tris-HCl，pH 8.0，2mL 1M MgCl2定容至1L)中，4℃搅拌透析过夜，中间换两次透析液。收集病毒，测定病毒滴度。利用感染细胞后提取病毒DNA定量方法检测病毒滴度。将约2×106个HeLa细胞/孔接种到6孔组织培养板上。一天后细胞覆盖率达到90-100％，用500μL无血清培养基稀释5μL病毒储备液感染细胞设两个复孔。37℃孵育3至6小时。除去培养基，1mL PBS/孔洗涤两次。加入500μL新鲜制备的NP-40裂解缓冲液(0.65％NP-40替代物(Calbiochem,Billerica,MA,USA)，150mM NaCl，10mM Tris，pH 8.0)裂解细胞。室温下孵育5-10分钟，上下吹打10-15次以帮助裂 解，2000×g离心3分钟使核沉淀，弃上清，加入1mL的NP-40裂解缓冲液，短暂涡旋，然后在2000×g下再进行3分钟的离心，弃上清，将细胞核悬浮于200μLPBS中。DNA分离和纯化，利用DNeasy Blood&Tissue kit(QIAgen,Valencia,CA,USA)提取DNA，用于后续定量反应。定量引物有L2 Forward Primer：TTGTGGTTCTTGCAGATATGGC(SEQ ID NO：19)；L2Reverse Primer：TCGGAATCCCGGCACC(SEQ ID NO：20)。
制备标准曲线：PCR L2目的序列构建到T载体上，将质粒稀释到1×109到1×102copy/μL作为标准序列。稀释公式为[Plasmid concentration(μg/μL)×Avogadro’s number×10-6]/[number of nucleotides×1bp molecular weight]＝plasmid copies/μL
6、细胞实验
检测腺病毒对不同细胞的识别与杀伤能力：Chang细胞1×104细胞/孔，HepG2、Huh7细胞5×104细胞/孔铺96孔板，腺病毒Ad vehicle以102到10-4十倍稀释七个梯度MOI(Multiplicity of Infection，感染复数)感染这三种细胞系，持续观察六天后利用MTS方法检测细胞存活率。
7、MTS检测
MTS法检测细胞数：在96孔板培养的待测细胞中每孔加20μL MTS/PMS混合液(Promega公司)，混匀后继续37℃培养1至4小时。显色后摇晃培养板10秒钟，混匀颜色。使用酶标仪检测波长490nm的吸光度(OD值)。绘制标准曲线，可以检测目的细胞的细胞数量。
8、细胞因子检测实验
细胞因子的生物活性可以通过因子依赖型细胞的增殖检测。分别使用CTLL-2，FDC-P1，TF-1细胞系检测IL-2，mGM-CSF，hGM-CSF的生物活性。收集待测因子依赖性的细胞，将1×104待测细胞加入96孔板后，随后加入含有待测细胞因子的样品，37℃恒温培养24小时或48小时之后，使用MTS方法检测细胞增殖，绘制剂量-增殖标准曲线，对比计算得出样品中对应细胞因子的活性水平。
9、免疫共沉淀实验
免疫共沉淀：收取待测细胞，使用适量蛋白裂解液(碧云天公司)裂解细胞，离心后去除细胞碎片。取少量裂解液以备Western blot分析，剩余裂解液将加入10-30μL偶联相应的标签抗体的protein A/G-agarose beads(Thermo Fisher Scientific公司)加入到细胞裂解液中，4℃缓慢摇晃孵育过夜。孵育后离心去除上清，将沉降后的珠子洗三遍后，使用洗脱液将抗体和捕获的蛋白从agarose beads上洗脱后，加入SDS-PAGE上样缓冲液(上海生工公司)煮沸后通过Western blot检测相关蛋白的富集情况。
10、动物实验
实验动物采购自华阜康或维通利华公司，肿瘤模型采用6周龄的雄性或者雌性小鼠，均饲养在SPF的环境中。人源肿瘤模型使用裸鼠构建，将1×107HepG2人肝癌细胞混合基质胶(BD公司)皮下注射，接种在裸鼠右侧腹部，每三天通过游标卡尺测量皮下瘤块的大小。鼠源肿瘤模型使用C57BL/6J野生型小鼠构建，将1×106Hepa1-6小鼠肝癌细胞皮下接种在小鼠的右侧腹部，每三天通过游标卡尺测量皮下瘤块的大小。待肿瘤体积达到100mm3后，给予溶瘤腺病毒或者对照治疗。根据动物实验伦理的要求，当肿瘤大小达到直径15mm后，将荷瘤小鼠安乐处死。小鼠肿瘤的体积通过近似公式(体积＝0.5×长×宽2)计算得出。
实验结果
1、利用合成生物学手段构建双抑制型开关响应多种生物标志物区分癌症细胞和非癌症细胞。
近些年的相关研究表明，溶瘤病毒是治疗癌症的十分有效的手段，其中溶瘤腺病毒已经开展很多临床研究。根据已有的临床研究结果表明，溶瘤腺病毒存在比较高比例的泄露现象，所以如何提高溶瘤腺病毒在临床阶段的安全性是一个很重要的问题。根据对已有的溶瘤腺病毒的分析发现，多数溶瘤腺病毒的调控依赖于单一的生物标志物，例如肿瘤特异性的启动子调控腺病毒复制的关键基因E1的表达。但单一的生物标志物存在比较高的泄露机率。所以在本实施例中发明人设计了一个双抑制型开关系统响应多种生物标志物，在多水平调控腺病毒E1A基因的表达，提高溶瘤腺病毒的识别特异性并进而增强安全性。
如图3所示，双抑制型开关系统的特点如下：肿瘤特异性启动子(pC,cancer specific promoter)调控调控双抑制型开关系统的总激活子(Activator)。激活子作用于激活子启动子(pAct,activator response promoter)调控下游基因的表达。双抑制系统的一侧表达腺病毒包装相关的E1A基因，效应因子(Effector)和抑制子a(Rep-a,repressor a)以及响应非肿瘤细胞中高表达的微小RNA a(miRNA a)的靶向序列。该侧的启动子两边有另一侧表达的抑制子b的识别序列(Tb,target of repressor b)和响应肿瘤细胞中高表达的微小RNA b(miRNA b)的靶向序列。双抑制系统的另一侧表达抑制子b(Rep-b,repressor b)。该侧的启动子两边有另一侧表达的抑制子a的识别序列(Ta,target of repressor a)。在肿瘤细胞中，pC启动整个系统的表达，由于在肿瘤细胞中miRNA b高表达同时miRNA a低表达，抑制子b被RNA干扰作用降解，解除了抑制子b对另一侧基因表达的抑制作用。同时抑制子a高表达可以进一步抑制抑制子b的表达，使双抑制型开关系统快速高效地翻转到E1A表达的状态，启动腺病毒的包装和效应因子的表达。相反地，当双抑制开关进入非肿瘤细胞内时，通过肿瘤特异性启动子，微小RNA标志物和逻辑线路的共同作用，无法表达E1A和效应因子，有效的关闭了腺病毒的包装过程，确保了该逻辑线路的特异性和安全性。所以这种开关可以响应多种输入信号在转录水平和转录后水平区分目的与非目的细胞类型。另外，在本系 统中发明人只表达了E1A基因，去除了E1B基因，从功能缺陷型互补的角度提高了溶瘤病毒目的细胞的区分能力。所以发明人设计的系统从多水平多层次区分目的细胞，可以有效提高腺病毒的安全性。
2、验证分析区分肝癌细胞和非肝癌细胞的生物标志物
2.1构建肝癌特异性的甲胎蛋白(AFP)启动子。
甲胎蛋白(α-fetoprotein，αFP或AFP)主要在胎肝中合成，在正常的成人体内没有AFP表达，但当肝细胞发生癌变时，AFP重新表达，而且随着病情恶化AFP在血清中的含量会急剧增加。目前甲胎蛋白是诊断原发性肝癌的一个特异性临床指标。所以在本实施例中发明人选择了甲胎蛋白启动子作为一个区分肝癌细胞和非肝癌细胞的启动子标志物。
发明人进一步通过实验检测AFP的特异性表达。首先通过qPCR检测了人AFP基因在一系列非肝癌和肝癌细胞中的表达。如图4所示提取Chang、HepG2、Huh7、PLC、Hep3B和Hepa1-6细胞的RNA。通过定量PCR检测AFP基因在这些细胞中的表达水平。Chang是人的正常的肝细胞，HepG2、Huh7、PLC、Hep3B是人的肝癌细胞。Hepa1-6是小鼠的肝癌细胞。人的AFP基因在HepG2和Huh7细胞系中高表达，在PLC和Hep3B细胞系中表达较低。在小鼠的肝癌细胞Hepa1-6中也检测到了轻微的表达。这些结果表明，AFP特异性的表达在肝癌细胞系中，可以作为发明人的启动子标志物。
目前被广泛应用的AFP启动子由增强子(enhancer)和启动子(promoter)两部分组成。增强子中包含两段激活启动子的序列DA(domain A)和DB(domain B)。为了提高启动子的效率，发明人将启动子区的-119位的G突变为A。另外，发明人对增强子区做了不同的截断实验。如图5所示AFPI包含了AFP的1.8K的增强子序列(序列如SEQ ID NO：21所示)；AFPII包含了激活区域A(DA)，激活区域B(DB)以及两个激活区域之间的序列(序列如SEQ ID NO：22所示)；AFPIII只包含了激活区域A(DA)和激活区域B(DB)(序列如SEQ ID NO：23所示)；AFPIV只包含激活区域A(DA)(序列如SEQ ID NO：24所示)；AFPV只包含激活区域B(DB)(序列如SEQ ID NO：25所示)。将这五种AFP启动子报告系统质粒瞬转入正常肝细胞Chang和肝癌细胞HepG2中检测AFP启动子的表达效率和特异性。从结果来看(如图6所示)五种AFP启动子在肝癌细胞系中都有很高的表达水平，甚至超过了常表达的CMV启动子。从特异性角度分析，在Chang中除了AFPII的报告基因有轻微表达，其它启动子都没有明显泄露。综合考虑插入序列的长度，启动子的效率和特异性发明人在后续的实验中选择了AFP III启动子调控发明人的双抑制型开关。
Figure PCTCN2017096043-appb-000041
Figure PCTCN2017096043-appb-000042
Figure PCTCN2017096043-appb-000043
Figure PCTCN2017096043-appb-000044
2.2构建微小RNA(microRNA)荧光质粒报告系统，检测不同细胞系中的表达水平。
现有的研究结果表明，随着肿瘤的发生发展，微小RNA(microRNA)的表达水平会发生很大变化。如表1所示，在不同的癌症细胞中存在很多高表达的microRNA。通过对已有的研究结果进行分析，发明人将miR199a作为表征正常肝细胞在正常肝细胞中高表达的microRNA标志物，相应地发明人将miR21和miR122作为表征肝癌细胞在肝癌细胞中高表达的microRNA标志物。首先，发明人设计构建了microRNA荧光质粒报告系统，如图7 所示，发明人将microRNA的靶标序列插入常表达的EYFP的3’非翻译区，以另一个常表达的EBFP作为对照荧光。共转入不同的细胞系检测靶标microRNA在目标细胞系中的表达。实验结果如图8所示，与肝癌细胞系相比miR199a在正常肝细胞中高表达；而miR21在肝癌细胞系中的表达水平显著升高，在发明人设计的双抑制开关系统中可以作为区分正常细胞与肝癌细胞的有效microRNA标志物。
2.3双抑制型开关可以有效的响应microRNA输入信号，在不同细胞系中稳定的实现功能。
由于溶瘤腺病毒的包装容量有限，最多只能包装8K外源基因，所以发明人再构建本实施例时尽量选取了短小有效的抑制元件。在本实施例中发明人选取了LacI和tetR-KRAB。如图9所示，发明人构建了两种逻辑线路：在tetR-KRAB边是否共表达EYFP基因。Switch I(SI)不表达EYFP，Switch II(SII)表达EYFP。发明人将两种开关分别瞬转入HEK293中，同时共转shRNA-FF4和shRNA-FF5。如图10所示，发明人的两种开关在不同的输入信号下都可以高效的翻转。但相比于SII，SI在LacI侧表达水平更高，但相应地在tetR-KRAB一侧泄露水平也会相较于SII要高一些。但综合考虑发明人的开关系统可以响应多种输入信号和高效的在目的细胞中表达E1基因，在后续实验中，发明人选择了SI系统作为骨架构建发明人荷载不同效应因子的溶瘤腺病毒。基于实验室之前的结果发明人检测了不同激活子对双向抑制型开关的控制效果。如图11所示发明人检测了Gal4VP16、Gal4esn、dCas9-VP64与rtTA的调控效率，实验结果显示基因线路可以有效地响应不同的激活子，所以这种基因线路具有良好的稳定性。
2.4利用CGGA的方法构建溶瘤腺病毒
大量的临床信息表明，肿瘤标志物在不同个体间的表达存在很大差异。所以针对于不同的个体发明人可选用的标志物很可能不同。为了实现对病人个体的精准医疗，发明人需要制备多种荷载有不同生物标志物的溶瘤腺病毒。然而利用传统的酶切连接的方法快速构建这些溶瘤病毒是一件十分困难的事。在本实施例中发明人设计构建了三级库利用CGGA(Cascade Golden-Gate Gateway/Gibson Assemble method)的方法实现了对溶瘤腺病毒的快速改装。首先如图12所示，将区分正常细胞与癌症细胞的相关标志元件如肿瘤特异性启动子，逻辑线路构建相关的抑制元件，区分癌症细胞的微小RNA的识别序列构建到一级载体质粒上，构成一级元件库。元件两端设计有EI(一种识别位点与切割位点不同的限制性内切酶例如Esp3I与BsaI)的识别位点，用于通过Golden Gate的方式将多个一级元件构建到二级表达系统上。构建表达系统库，从一级库中选取多个一级元件通过Golden Gate的方式将他们组装成三个表达系统，包括肿瘤特异性启动子系统，第一调控元件所介导的抑制系统和第二调控元件所介导的抑制系统。然后，如图13所示，组装逻辑线路，通过Golden Gate的方式随机的将三种表达系统组装起来形成完整的逻辑线路。逻辑线路两端设计有用于下 一步实验的Gibson同源序列或者Gateway识别位点(target sequence)。最后如图14所示，通过Gibson或者Gateway的方式将逻辑线路构建到已经去除了E1和部分E3的腺病毒载体上。利用PacI酶切将改造后的腺病毒载体线性化。然后转染入特定细胞系中包装腺病毒颗粒。这样发明人可以快速的替换掉一种到多种肿瘤生物标志物。
2.5在细胞水平检溶瘤腺病毒可以有效的杀伤肝癌细胞
为了检测发明人构建的双抑制型开关系统在溶瘤腺病毒里是否可以有效的区分杀伤肝癌细胞。发明人设计了溶瘤腺病毒的体外细胞杀伤实验。以不同的感染复数感染不同的细胞系Chang是正常的肝细胞，Huh7和HepG2是人的肝癌细胞系。加入病毒培养六天后利用MTS方法检测细胞的存活率。如图15所示，发现在感染复数为100，10，1的时候，肝癌细胞发生明显的细胞毒性反应大量死亡，在感染复数为0.1时有轻微的细胞死亡。相应的，在感染复数为10及以下时Chang细胞生长状态正常没有死亡现象。在感染复数为100时，细胞有轻微的细胞毒反应，同时细胞体积变大，细胞生长变慢。这一数据表明在特定的感染复数内，发明人包装的腺病毒可以有效地区分杀伤肝癌细胞，同时不影响正常肝细胞的生长与增殖。
2.6体外检测溶瘤腺病毒携带的效应因子的表达水平
由于目前临床上使用的腺病毒载体为人的Ad2或Ad5型腺病毒，在自然界中这些病毒广泛存在，所以人体内已经存在了拮抗这些腺病毒的中和抗体。所以只依靠腺病毒的溶瘤作用治疗肿瘤，临床数据显示治疗效果并不好。所以在本实施例中发明人利用溶瘤腺病毒作为载体将效应因子基因携带到肿瘤的靶标位置引起机体系统性的免疫反应。如图16所示，发明人首先将这些效应因子构建到常表达的质粒载体上，通过瞬转的方式检测这些效应因子的活性。结果表明发明人表达的效应因子基因可以有效地产生具有生物学功能的免疫因子和免疫检验点基因的抑制子。发明人将这些效应因子基因构建到溶瘤腺病毒载体上，包装纯化出荷载这些效应因子的溶瘤腺病毒。感染肝癌细胞HepG2后，发明人吸取上清检测了上清中免疫因子的活性。
同时发明人检测了腺病毒载体携带的细胞效应因子的活性，如图17所示，病毒携带的细胞效应因子随时间有效增强。三种细胞因子的检测方法如下所述，图17a 12孔板接种3×106HepG2细胞，分别用synOV-EBFP和synOV-mGM-SCF以10的感染复数感染HepG2.分别在1，2，3，4天收取上清,利用ELISA试剂盒检测上清中小鼠GM-CSF的含量。图17b12孔板接种3×106HepG2细胞，分别用synOV-EBFP和synOV-hIL-2以10的感染复数感染HepG2.分别在1，2，3，4天收取上清,将上清加入IL-2依赖型细胞的培养基中，通过MTS检测上清中IL-2的含量。图17c 12孔板接种3×106HepG2细胞，分别用synOV-EBFP，synOV-anti-PD-1scFv和synOV-anti-PD-L1scFv以10的感染复数感染HepG2。分别在1，2， 3，4天收取上清。分离小鼠脾细胞，用2ug/mL抗小鼠CD3抗体激活T细胞，同时以1：10的稀释比例将上述收取的上清加入脾细胞系统。48小时后用ELISA检测脾细胞中IFN-γ的产量。
2.7利用动物实验检测溶瘤腺病毒的治疗效果
如图18所示，将1×107HepG2,Huh7，Hepa1-6肝癌细胞皮下接种在裸鼠右侧腹部，每三天测量皮下瘤块的大小。HepG2和Huh7是人的肝癌细胞，Hepa1-6是小鼠的肝癌细胞待肿瘤体积达到100mm3后，随机分为两组，治疗组瘤内注射1×109溶瘤腺病毒，以注射PBS作为对照组。持续观测治疗后肿瘤体积的变化，可以发现在溶瘤腺病毒注射后，PBS对照组的肿瘤持续生长，体积持续增大，而治疗组的肿瘤生长受到了明显的抑制，证明溶瘤腺病毒通过对肿瘤细胞的裂解作用，可以抑制体内肿瘤体积的增长，对已经成型的肿瘤有显著的治疗效果。在治疗小鼠肝癌时发明人加入了另一种对照病毒以第二代腺病毒载体负载的GFP基因病毒(Ad-GFP),这种病毒是非溶瘤性病毒，从实验结果可以发现PBS和AdGFP治疗组的肿瘤持续生长，溶瘤病毒治疗组可以在一定程度上控制肿瘤的生长，但并没有有效控制肿瘤生长，可能是因为Hepa1-6的生长速度比HepG2和Huh7快，同时在Hepa1-6中AFP的表达水平比较低等原因造成的。因此发明人在后续试验中利用负载免疫效应因子的溶瘤腺病毒继续治疗Hepa1-6肿瘤模型，取得了十分有效结果。
在荷瘤小鼠接受溶瘤腺病毒治疗2周或者一个月后，取小鼠各器官的组织样品，匀浆后提取病毒DNA。通过qPCR方法检测病毒滴度，发现在病毒感染之后，病毒仅在在肿瘤细胞里复制，并未扩散到其他组织内。这证明在体内溶瘤腺病毒有很强的特异性，对正常组织的细胞是安全的。
进一步检测发明人的溶瘤腺病毒对具有免疫系统的C57小鼠模型的治疗效果。如图19所示，将1×106Hepa1-6小鼠肝癌细胞皮下接种在野生型C57BL/6J小鼠的右侧腹部，待肿瘤体积达到100mm3后，每组分别瘤内注射1×109表达不同细胞因子溶瘤腺病毒，或者注射PBS作为阴性对照。观测经过治疗后肿瘤体积的变化，并通过Kaplan-Meier法分析荷瘤小鼠的生存时间。实验结果证明，在免疫系统健全的小鼠体内，溶瘤腺病毒仍然可以抑制肿瘤的生长，显著延长荷瘤小鼠的生存时间，部分荷瘤小鼠经治疗后肿瘤完全消失，达到了治愈的效果。在溶瘤腺病毒治疗一个月后，在存活的小鼠的左侧腹部再次接种1x106Hepa1-6小鼠肝癌细胞，结果发现，在同一只小鼠的不同部位接种与初次接种相同的肿瘤细胞系，无法长成瘤体。这证明经过溶瘤病毒治疗的小鼠，机体产生了对肿瘤细胞的特异性免疫力，可以抵抗同一种肿瘤细胞在机体内再次生长。
取荷瘤小鼠的肿瘤，固定、切片后通过HE染色法检测肿瘤组织的淋巴细胞浸润情况。可以 发现经过溶瘤腺病毒治疗的小鼠肿瘤组织的淋巴细胞浸润比PBS对照组明显增加，证明溶瘤腺病毒的治疗在肿瘤组织中招募了更多的淋巴细胞。分离纯化肿瘤中浸润的淋巴细胞，通过流式细胞术检测小鼠肿瘤浸润T细胞的表型，发现通过溶瘤病毒治疗的小鼠肿瘤内部浸润的Ki-67+的T细胞的比例更高，证明溶瘤腺病毒治疗促进了肿瘤内部T细胞的增殖，引发了更强的免疫应答。把肿瘤浸润的淋巴细胞加入含有PMA(20ng/mL)和Ionomycin(1μg/mlL)的RPMI 1640培养基，在Brefedlin A存在的情况下，37摄氏度培养4小时。细胞固定染色后，通过流式细胞仪检测CD4+，CD8+T细胞中γ干扰素的表达情况。发现在溶瘤腺病毒治疗组的小鼠肿瘤内，T细胞的γ-干扰素的表达更高，改变了肿瘤内部的免疫微环境，促进了肿瘤细胞的清除，如图20(a，b)所示。
溶瘤腺病毒治疗引起全身性的抗肿瘤免疫应答
在C57BL/6小鼠腹部左侧和右侧的皮下都接种小鼠Hepa-1-6肿瘤，成瘤后，右侧瘤内分别注射表达细胞因子hIL-2，mGM-CSF，anti-PD-1scFv的溶瘤病毒，PBS对照和AdGFP对照，持续测量的肿瘤生长。治疗14天后，取荷瘤小鼠的两侧肿瘤。分离纯化肿瘤中浸润的淋巴细胞，通过流式细胞术检测小鼠肿瘤浸润T细胞的表型。发现不仅在右侧注射病毒的肿瘤内，在左侧未经注射病毒的肿瘤内，与对照组相比，Ki-67+的T细胞的比例更高，表达γ-干扰素的T细胞的比例更高。证明溶瘤腺病毒的治疗所引起的免疫反应不仅可以作用于注射侧的肿瘤，也作用于远端的肿瘤。这提示溶瘤腺病毒的肿瘤治疗效果不仅仅是作用于注射处的瘤体，对远端的转移灶也有一定的治疗效果。
同时为了检测溶瘤腺病毒的治疗是否是存活小鼠体内产生有效的系统免疫反应。发明人重新在治疗后的小鼠皮下接种Hepa1-6，观测小鼠的生长状况发现所有小鼠的排斥率为100％，如图21所示
2.8对溶瘤腺病毒的治疗过程进行建模分析
将溶瘤腺病毒的治疗情境抽象为肿瘤-病毒-免疫系统，如图22，包含未受侵染的肿瘤细胞、受侵染的肿瘤细胞、游离病毒、受溶瘤腺病毒抗原激活的免疫细胞与受肿瘤抗原激活的免疫细胞五种组分，分别用S、I、V、ZV与ZT表示。模型假设未受侵染的肿瘤细胞生长符合广义逻辑斯蒂增长模型，其中γ为增长速率系数，K为环境容量，ε为非线性系数。未受侵染的肿瘤细胞可以被游离病毒侵染，转变为受侵染的肿瘤细胞，侵染速率为κ。受侵染的肿瘤细胞将裂解并释放更多病毒，裂解速率系数为δ，游离病毒释放速率系数为α。游离病毒在环境中会自然衰减，衰减速率系数为ω。ZV会受溶瘤腺病毒与被病毒侵染的肿瘤细胞表面抗原激活而增长，速率系数分别为cV1与cV2，而ZV会对受侵染的肿瘤细胞产生抑制作用，速率系数为pV。ZT会受肿瘤细胞裂解后释放的抗原激活而增长，速率系数为cT，而ZT会对受侵染与未受侵染的肿瘤细胞产生抑制作用，速率系数为pT。ZV与ZT在环境中会 自然衰减，速率系数为β。
在不考虑免疫系统的情况下，随病毒复制速度增加，系统终状态将由收敛转为振荡。仿真图所示为，在不同的初始病毒滴度与初始肿瘤大小条件下，病毒复制速度较低与较高时，肿瘤-病毒系统随时间变化至终状态的过程中，肿瘤体积的最小值的变化情况。从仿真结果可以看出，增加病毒初始滴度有助于控制肿瘤大小。在实际应用中，可结合初始肿瘤大小与给药剂量限制，选择合适的初始给药剂量，从而在预测的时间点将肿瘤清除或辅以其它疗法，结果如图23所示。
在免疫细胞对肿瘤与病毒不同的抑制作用强度影响下，系统在仿真时间内终状态的肿瘤大小。从仿真结果可以看出，系统表现出趋于完全清除肿瘤、肿瘤生长至饱和、肿瘤体积稳定及肿瘤体积出现振荡几种动态行为。总体上，免疫细胞对肿瘤的抑制作用越强，系统越趋于清除肿瘤，而免疫细胞对病毒的抑制作用越强，系统越趋于对肿瘤失去控制，而对于肿瘤稳定或振荡，系统关于两种免疫作用的趋势表现出一些非单调变化，结果如图24所示。
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。
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。

Claims (40)

  1. 一种表达系统，其特征在于，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；
    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；以及
    选自下列的至少之一：
    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；
    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。
  2. 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肿瘤细胞特异性启动子，所述肿瘤细胞特异性启动子为选自甲胎蛋白特异性启动子，Survivin基因启动子,人端粒酶逆转录酶基因启动子,胆囊收缩素A受体基因启动子，癌胚抗原启动子，原癌基因人类表皮生长因子受体2启动子，前列腺素内氧化酶还原酶2启动子，趋化因子受体-4，E2F-1基因启动子，黏蛋白启动子，前列腺特异抗原，人酪氨酸酶相关蛋白1，酪氨酸酶启动子至少之一。
  3. 根据权利要求1所述的表达系统，其特征在于，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。
  4. 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5×UAS,7×tetO以及dCas9的靶标序列至少之一。
  5. 根据权利要求1所述的表达系统，其特征在于，所述第一启动子与所述第二启动子分别独立地选自miniCMV、TATAbox。
  6. 根据权利要求1所述的表达系统，其特征在于，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、KRAB、tetR-KRAB、dCas9-KRAB至少之一。
  7. 根据权利要求6所述的表达系统，其特征在于，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、dCas9的靶标序列至少之一。
  8. 根据权利要求7所述的表达系统，其特征在于，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。
  9. 根据权利要求6所述的表达系统，其特征在于，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。
  10. 根据权利要求1所述的表达系统，其特征在于，所述第五核酸分子和第九核酸分子的至少之一进一步包括编码目的蛋白的序列。
  11. 根据权利要求10所述的表达系统，其特征在于，所述第五核酸分子包括编码所述目的蛋白的序列，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。
  12. 根据权利要求的11所述的表达系统，其特征在于，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。
  13. 根据权利要求11所述的表达系统，其特征在于，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。
  14. 根据权利要求10所述的表达系统，其特征在于，所述目标蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目标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。
  15. 根据权利要求1所述的表达系统，其特征在于，所述第九核酸分子与所述第十核酸分子分别独立地通过下列方式抑制所述第一调控蛋白或所述第二调控蛋白的表达：借助RNA干扰。
  16. 根据权利要求15所述的表达系统，其特征在于，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。
  17. 根据权利要求16所述的表达系统，其特征在于，所述第一microRNA包括选自下列的至少之一：
    miR199a、miR95、miR125、miR25b、Let-7、miR143、miR145以及miR200C。
  18. 根据权利要求16所述的表达系统，其特征在于，所述第二microRNA包括选自下列的至少之一：
    miR21、miR223、miR224、miR221、miR18、miR214、miR146a以及miR1792。
  19. 根据权利要求1所述的表达系统，其特征在于，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可选的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可选的所述第十核酸分子负载在第三表达载体上。
  20. 根据权利要求19所述的表达系统，其特征在于，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：
    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。
  21. 根据权利要求20所述的表达系统，其特征在于，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。
  22. 根据权利要求19所述的表达系统，其特征在于，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。
  23. 根据权利要求22所述的表达系统，其特征在于，所述同一个载体为腺病毒载体。
  24. 根据权利要求19所述的表达系统，其特征在于，所述第一表达载体包括：
    从5’端到3’端依次为BsaI，AFP III，Gal4VP16以及BsaI，
    任选地，第一表达载体携带具有SEQ ID NO：1所示核苷酸序列的核酸。
  25. 根据权利要求19所述的表达系统，其特征在于，所述第二表达载体包括：
    从5’端到3’端依次为BsaI，5×UAS，tetO，miniCMV，tetO，E1A，2A，免疫效应因子，LacI，microRNA199a特异识别序列以及BsaI，
    任选地，第二表达载体携带具有SEQ ID NO：2～7任一项所示核苷酸序列的核酸。
  26. 根据权利要求19所述的表达系统，其特征在于，所述第三表达载体包括：
    从5’端到3’端依次为BsaI，5×UAS，LacO，miniCMV，LacO，tetR-KRAB，microRNA21特异识别序列以及BsaI，
    任选地，所述第三表达载体携带具有SEQ ID NO：8所示核苷酸序列的核酸。
  27. 根据权利要求23所述的表达系统，其特征在于，所述腺病毒包括：
    从5’端到3’端依次为第一反向末端重复序列,包装信号，AFPIII，Gal4VP16，5×UAS，tetO，miniCMV，tetO，E1A，2A，免疫效应因子，2A，LacI，microRNA199a特异性识别序列，5×UAS，LacO，miniCMV，LacO，tetR-KRAB，microRNA21特异性识别序列,腺病毒E2基因区，E3基因区，E4基因区以及第二反向末端重复序列，
    任选地，所述腺病毒载体携带具有SEQ ID NO：9～14所示核苷酸序列的核酸。
  28. 根据权利要求27所述的表达系统，其特征在于，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。
  29. 一种重组病毒，其特征在于，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，所述肿瘤细胞特异性启动子为甲胎蛋白特异性启动子；
    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，所述第一识别序列为5×UAS；
    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，
    所述目标蛋白与所述第一调控蛋白是以融合蛋白的形式表达的，并且所述目标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，所述第二识别序列为5×UAS；
    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。
  30. 根据权利要求29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。
  31. 根据权利要求29所述的重组病毒，其特征在于，所述重组病毒为腺病毒。
  32. 根据权利要求29所述的重组病毒，其特征在于，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。
  33. 一种重组细胞，其特征在于，含有权利要求1～28所述的表达系统。
  34. 根据权利要求33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。
  35. 权利要求1～28任一项所述的表达系统、权利要求29～32任一项所述的重组病毒、权利要求33～34任一项所述的重组细胞在制备药物中的用途，所述药物用于治疗癌症。
  36. 根据权利要求35所述的用途，其特征在于，所述癌症包括肝癌、肺癌、结直肠癌，黑色素瘤、乳腺癌或前列腺癌。
  37. 一种利用表达系统表达目的蛋白的方法，所述表达系统为权利要求1～28任一项所述的表达系统，其特征在于，所述方法包括：
    (1)使所述第五核酸分子包含编码所述目的蛋白的核酸序列；
    (2)使所述第十核酸分子抑制所述第二调控蛋白的表达，以便表达所述目的蛋白。
  38. 根据权利要求37所述的方法，其特征在于，所述表达是在细胞中进行的。
  39. 根据权利要求37所述的方法，其特征在于，所述第十核酸分子含有被第二microRNA特异性识别的核酸序列，在步骤(2)中包括使所述第二microRNA与所述第十核酸分子接触。
  40. 一种利用表达系统表达目的蛋白的方法，所述表达系统为权利要求1～28任一项所述的表达系统，其特征在于，所述方法包括：
    (1)使所述第八核酸分子包含编码第一目的蛋白的核酸序列，使所述第五核酸分子包含编码第二目的蛋白的核酸序列；
    (2)通过下列之一表达所述第一目的蛋白或所述第二目的蛋白：
    使所述第九核酸分子抑制所述第一调控蛋白的表达，以便表达所述第一目的蛋白；
    使所述第十核酸分子抑制所述第二调控蛋白的表达，以便表达所述第二目的蛋白。
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