CN116814688A

CN116814688A - Vector carrying bispecific promoter, construction method and application thereof

Info

Publication number: CN116814688A
Application number: CN202310794540.0A
Authority: CN
Inventors: 宋灿; 王振宇; 冉杰; 田甜; 于杨; 郭平; 顾雨春; 吴理达
Original assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2023-09-29
Anticipated expiration: 2043-06-30
Also published as: CN116814688B

Abstract

The invention discloses a carrier carrying a bispecific promoter, a construction method and application thereof, and provides a carrier of the bispecific promoter, a construction method of the carrier of the bispecific promoter, a host cell comprising the carrier of the bispecific promoter, a virus and a preparation method thereof, and a related pharmaceutical composition which can be specifically expressed in specific tumor cells to realize accurate killing of the tumor cells, and meanwhile, the carrier of the bispecific promoter in the invention is not expressed or is rarely expressed in non-tumor cells, so that the probability of accidental injury of normal cells is reduced, the safety is improved, and the pharmaceutical composition has wide market application prospect.

Description

Vector carrying bispecific promoter, construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a vector carrying a bispecific promoter, a construction method and application thereof.

Background

Gene medicine using adeno-associated virus (AAV) as carrier has the characteristics of durable gene expression and low immunogenicity, and has been widely used in the fields of monogenic genetic diseases, ophthalmic diseases, nervous system diseases, etc. However, AAV has weak infection specificity on tumor cells, so that normal cells can be infected while tumor cells are infected, and the application of AAV in the field of tumor treatment is limited.

The Tumor specific promoter (Tumor-specific promoter) holds promise for the application of AAV in the Tumor field. Tumor specific promoters refer to promoter sequences that are specifically expressed in tumor cells. These promoters have higher activity than normal cells or are activated only in tumor cells. The application of the tumor specific promoter can achieve various purposes. First, they can be used for tumor gene therapy, and by combining a therapeutic gene with a tumor specific promoter, the therapeutic gene is expressed only in tumor cells, thereby improving the specificity and safety of the therapy. Second, tumor specific promoters can also be used in tumor immunotherapy to promote immune cells in tumor-specific anti-tumor responses locally by combining immune-related genes with tumor specific promoters. In addition, the tumor specific promoter can also be used for tumor diagnosis, and the visualization of tumor cells or the development of molecular probes can be realized by combining a reporter gene with the tumor specific promoter.

Disclosure of Invention

The invention provides a carrier carrying a dual-specificity promoter, a construction method and application thereof, which have killing function when two specific promoters are simultaneously started, and improve the accuracy and safety of tumor treatment in order to make up for the defects of the prior art and solve the specific problems in the prior art.

The invention provides a vector of a bispecific promoter, wherein the bispecific promoter is two specific promoters.

The carrier comprises: the bispecific promoters are linked to the genes of interest, respectively.

The gene of interest comprises at least one of the following combinations: a combination of Casp1act and GSDMD, a combination of GSDMD C and GSDMD N.

The two components of each combination of the target genes can be respectively called target gene 1 and target gene 2.

The term "vector" includes biological or chemical parts of a nucleic acid sequence that can be introduced into an appropriate host cell for replication or expression of the nucleic acid sequence. Vectors include non-viral vectors and viral vectors. In some embodiments of the invention, the non-viral vector may be selected from the group consisting of nanoparticles, novel biological materials, naked DNA, phage, transposons, plasmids, cosmids, artificial chromosomes. Plasmids, other cloning and expression vectors, their nature, and methods of construction that can be used in accordance with the present invention will be apparent to those skilled in the art.

Further, the two promoters of the bispecific promoter may be referred to as specific promoter 1, specific promoter 2, respectively.

Further, the structure of the vector of the bispecific promoter comprises a specific promoter 1, a target gene 1, a specific promoter 2 and a target gene 2 which are connected in sequence.

Further, the specific promoter and the target gene are connected with a special sticky end to ensure the accuracy of connection.

Further, the specific promoter is a cancer-associated specific promoter.

Further, the cancer includes squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, peritoneal cancer, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic cancer, anal cancer, penile cancer, scrotal cancer, esophageal cancer, biliary tract tumor, and cancer of the head and neck.

Further, the two promoters corresponding to the bispecific promoter are any two of PCA3, PEG3, human telomerase reverse transcriptase promoter, survivin promoter, midkine promoter, carcinoembryonic antigen promoter, MUC-1 promoter and probasin gene promoter.

Further, the vectors include plasmids, lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, linear polynucleotides.

Further, the vector includes an AAV viral core plasmid.

Further, the vector of the bispecific promoter comprises a vector with the structure AAV-PCA3-Casp1act-PEG 3-GSDMD.

Further, the vector of the bispecific promoter comprises a Casp1act nucleotide sequence as shown in SEQ ID NO. 1.

Further, the vector of the bispecific promoter comprises a GSDMD nucleotide sequence as shown in SEQ ID NO. 2.

Further, the vector of the bispecific promoter comprises the PCA3 nucleotide sequence as shown in SEQ ID NO. 3.

Further, the vector of the bispecific promoter comprises a PEG3 nucleotide sequence as shown in SEQ ID NO. 4.

Further, the vector structure of the bispecific promoter comprises the nucleotide sequences of SEQ ID NO. 3, SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO. 2 which are connected in sequence.

In certain embodiments, the structure of the vector of the bispecific promoter is AAV-PCA3-Casp1act-PEG3-GSDMD, which means that the PCA3 promoter, casp1act, PEG3 promoter and GSDMD are sequentially connected in AAV virus. In certain specific embodiments, the PCA3 promoter, casp1act, PEG3 promoter, GSDMD are linked in sequence in the AAV virus comprising the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:2 linked in sequence. In certain embodiments, the structure of the vector of the bispecific promoter is AAV-PEG3-GSDMD-PCA3-Casp 1act, meaning that PEG3 promoter, GSDMD, PCA3 promoter, casp1act are linked in sequence in an AAV virus. In certain specific embodiments, sequentially ligating a PEG3 promoter, GSDMD, PCA3 promoter, casp1act in the AAV virus comprises sequentially ligating the nucleotide sequences of SEQ ID NO. 4, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 1.

In certain embodiments, the vector may comprise other suitable "regulatory elements" or "regulatory sequences" in addition to the promoter, including but not limited to enhancers; a transcription factor; a transcription terminator; high efficiency RNA processing signals such as splicing and polyadenylation signals (polyA); sequences that stabilize cytoplasmic mRNA, such as the Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE); sequences that enhance translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and, if desired, sequences that enhance secretion of the encoded product. In certain embodiments, examples of the polyA include SV40, bovine growth hormone (bGH), and TK polyA. In certain embodiments, examples of such enhancers include the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, the LSP (TH binding globulin promoter/alpha 1-microglobulin/specific library protein (bikunin) enhancer), and other enhancers.

The invention also provides a construction method of the vector of the bispecific promoter, which is used for constructing the vector, and comprises the following steps:

obtaining a gene of interest comprising at least one of the following combinations: a combination of Casp1act and GSDMD, a combination of GSDMD C and GSDMD N;

obtaining a bispecific promoter;

and assembling the combination of the target genes and the bispecific promoter on a vector to obtain the vector.

Further, the two components of each combination of the genes of interest may be called the gene of interest 1 and the gene of interest 2, respectively.

Further, the two promoters of the bispecific promoter comprise any two of PCA3, PEG3, human telomerase reverse transcriptase promoter, survivin promoter, midkine promoter, carcinoembryonic antigen promoter, MUC-1 promoter and probasin gene promoter.

Further, the vector is an AAV viral core plasmid.

In certain embodiments, the AAV virus comprises an AAV viral core plasmid or an AAV viral core plasmid engineered plasmid, the structure of which comprises an AAV capsid and a vector genome packaged in the capsid.

The term "vector genome" refers to an engineered viral or non-viral core vector, generally referred to as a plasmid, lentiviral vector, retroviral vector, adenoviral vector, core genetic material in an adeno-associated viral vector, linear polynucleotide, and the like. The "vector genome" may be, under appropriate conditions, the core genetic material of a non-proviral vector or the core genetic material of a non-proviral vector. In certain specific embodiments, the vector genome may be a vector of the bispecific promoters described herein. In certain specific embodiments, the vector genome comprises a viral core plasmid or a viral core plasmid engineered plasmid.

In certain embodiments, an AAV viral vector is provided. An "AAV virus" is a dnase resistant viral particle comprising two elements, an AAV capsid and a vector genome comprising at least non-AAV coding sequences packaged within the AAV capsid. The source of AAV capsids can be any of several tens of naturally occurring and available adeno-associated viruses, as well as engineered AAV.

The term "AAV capsid" or "AAV vector intermediate" refers to an assembled AAV capsid lacking the desired genomic sequences packaged therein. It may also be referred to as an "empty" capsid. The capsid may contain no gene sequence of interest or only partially packaged genomic sequence insufficient to effect expression of the gene product. The "empty" capsids are nonfunctional to transfer the gene of interest to the host cell.

Further, the construction method comprises any one of the following steps:

a. amplifying the combination of the target genes, amplifying two promoters of the dual-specificity promoter, respectively connecting the target genes with the specificity promoter, and cloning the connecting object to the carrier to obtain the carrier comprising the dual-specificity promoter and the target genes;

b. amplifying the target gene and two promoters of the specific promoter, and then directly cloning the target gene and the two promoters on the vector to obtain the vector comprising the bispecific promoter and the target gene.

The invention also provides a host cell comprising the vector as described above.

Further, the host cells include prokaryotic cells and eukaryotic cells.

Further, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia.

Further, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells.

Further, the mammalian cells include fibroblasts, 293T cells.

In one embodiment, the host cell is a cell capable of engineering culture. The term "host cell" may refer to a packaging cell line in which the vector is produced from a production plasmid. In a certain embodiment, the term "host cell" may refer to any target cell in which transgene expression is desired. Thus, "host cell" refers to a prokaryotic or eukaryotic cell containing an exogenous or heterologous nucleic acid sequence that has been introduced into the cell by any means (e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA coated aggregates, viral infection, and protoplast fusion).

In a particular embodiment, the term "host cell" refers to a culture of cells of various mammalian species used for in vitro evaluation of the compositions described herein. In other embodiments herein, the term "host cell" refers to a cell used to produce and package a viral vector or recombinant virus. In another embodiment, the term "host cell" is intended to refer to a target cell of an individual treated in vivo for a disease or condition as described herein. In certain embodiments, the term "host cell" is a prostate cell.

The invention also provides a virus comprising the vector as described above.

The carrier generation process may comprise, for example, the following method steps: cell culture initiation, cell passage, cell inoculation, transfection of cells with plasmid DNA, replacement of the post-transfection medium with serum-free medium, and harvesting of the vector-containing cells and medium.

Further, the viruses include lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, poxviruses, papovaviruses, baculoviruses, papillomaviruses.

Further, the virus is an adeno-associated virus.

The invention also provides a preparation method of the virus, which comprises the following steps:

transfecting the vector as described above into a host cell; and amplifying and expressing the transfected host cells to obtain the virus.

Further, the virus is an adeno-associated virus.

Further, the means by which the vector is transformed into a host cell include electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, transfection, liposome delivery, membrane fusion techniques, high-speed DNA coated aggregates, viral infection, and protoplast fusion.

Further, the host cells include prokaryotic cells and eukaryotic cells.

Further, the mammalian cells include fibroblasts, 293T cells.

The invention also provides a pharmaceutical composition comprising the vector as described above, the host cell as described above, and the virus as described above.

Further, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary ingredients.

Further, the adjunct ingredients include solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, gums.

Further, the pharmaceutical composition is in the form of a lipid particle, liposome, vesicle, nanosphere or nanoparticle or the like.

In certain embodiments, the pharmaceutical composition is engineered into a non-viral vector, such as a plasmid or other genetic element, which is admixed with a carrier, diluent, excipient, and/or the like to form the pharmaceutical composition. In certain embodiments, the pharmaceutical composition is engineered into a viral vector that is mixed with a carrier, diluent, excipient, and/or the like to form the pharmaceutical composition. In certain embodiments, the pharmaceutical composition is engineered into a vector genome that is packaged into an AAV capsid to form an adeno-associated virus, which is mixed with a carrier, diluent, excipient, and/or the like to form the pharmaceutical composition. In certain embodiments, the vector genome may be packaged into different viruses.

The term "adjunct ingredients" encompasses any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, gums, and the like. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the compositions. By "pharmaceutically acceptable" is meant molecular entities and compositions that do not produce allergic or similar untoward reactions when administered to a host. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the desired organ (e.g., liver (optionally via hepatic artery), lung, heart, eye, kidney), oral, inhalation, intranasal, intrathecal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parental routes of administration. The routes of administration may be combined, as desired.

Any suitable route of administration (e.g., oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intravenous, intramuscular, intraperitoneal, and other parental routes) may be selected. Thus, the pharmaceutical compositions may be formulated for any suitable route of administration, for example, in the form of a liquid solution or suspension (e.g., for intravenous administration, for oral administration, etc.). Alternatively, the pharmaceutical composition may be in solid form (e.g., in the form of a tablet or capsule, e.g., for oral administration). In some embodiments, the pharmaceutical composition may be in the form of a powder, drops, spray, or the like.

The invention also provides the use of the vector as defined above, the method of construction as defined above, or the host cell as defined above in the preparation of a virus or plasmid.

Further, the virus is an adeno-associated virus.

The invention also provides the application of the vector, the construction method, the host cell, the virus or the preparation method in preparing medicines for treating tumors.

The terms "comprises," "comprising," "includes," "including," and variations thereof include other components, elements, integers, steps, and the like. In contrast, the term "consisting of … …" and variations thereof do not include other components, elements, integers, steps, etc.

The invention has the beneficial effects that:

the invention provides a vector of a double-specific promoter, which has the function of being capable of specifically expressing in tumor cells, and expressing two necessary target genes under the condition that two promoters are started, wherein the interaction of the two target genes plays a role in killing the tumor cells; meanwhile, the vector of the bispecific promoter is not expressed and rarely expressed in non-tumor cells, and if one promoter is abnormally expressed in the non-tumor cells, the vector does not play a role in killing cells, so that the probability of accidental injury of normal cells is reduced, the safety is improved, and the vector has a wide market application prospect.

Drawings

FIG. 1 is a graph of the expression of different promoters in Fibroblasts (FB);

FIG. 2 is a graph of the expression of different promoters in normal prostate cells (RWPE-1);

FIG. 3 is a graph of the expression of different promoters in prostate cancer cells (C4-2);

FIG. 4 is a graph showing the killing of different plasmid in prostate cancer cells (C4-2) cells;

FIG. 5 is a graph of killing by different plasmids in normal prostate cells (RWPE-1);

FIG. 6 is a graph of in vivo experimental effectiveness of AAV, wherein A is a graph of tumor effect after different plasmids are inoculated into a control group of mice and an experimental group of mice, and B is a graph of tumor volume after inoculation;

FIG. 7 is a graph showing the statistical weight of control mice and experimental mice after inoculation with different plasmids;

FIG. 8 is a biochemical test diagram of mouse blood, wherein A is a graph of transglutaminase, B is a graph of glutamic pyruvic transaminase, C is a graph of alkaline phosphatase, and D is a graph of glutamic oxaloacetic transaminase.

Detailed Description

The following describes the embodiments of the present invention in further detail with reference to examples.

Example 1 specific promoter verification

1. Experimental materials

(1) Prostate cancer cell C4-2, normal prostate cell Rwpe-1, fibroblast FB, 293T cell;

(2) PEI transfection reagent.

2. Experimental method

1) Vector construction

The SFFV-EGFP gene (control group), AAV-SFFV-Casp1act-GSDMD, PCA3-Casp1act-PEG3-GSDMD were synthesized by using EGFP as control, and ligated to AAV vector through cleavage sites 5'-PacI and 3' -EcoRI, respectively.

The nucleotide sequences of the elements are as follows:

casp1act nucleotide sequence (SEQ ID NO: 1):

ATGAACCCAGCTATGCCCACATCCTCAGGCTCAGAAGGGAATGTCAAGCTTTGCTCCCTAGAAGAAGCTCAAAGGATATGGAAACAAAAGTCGGCAGAGATTTATCCAATAATGGACAAGTCAAGCCGCACACGTCTTGCTCTCATTATCTGCAATGAAGAATTTGACAGTATTCCTAGAAGAACTGGAGCTGAGGTTGACATCACAGGCATGACAATGCTGCTACAAAATCTGGGGTACAGCGTAGATGTGAAAAAAAATCTCACTGCTTCGGACATGACTACAGAGCTGGAGGCATTTGCACACCGCCCAGAGCACAAGACCTCTGACAGCACGTTCCTGGTGTTCATGTCTCATGGTATTCGGGAAGGCATTTGTGGGAAGAAACACTCTGAGCAAGTCCCAGATATACTACAACTCAATGCAATCTTTAACATGTTGAATACCAAGAACTGCCCAAGTTTGAAGGACAAACCGAAGGTGATCATCATCCAGGCCTGCCGTGGTGACAGCCCTGGTGTGGTGTGGTTTAAAGATGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGCTATTAAGAAAGCCCACATAGAGAAGGATTTTATCGCTTTCTGCTCTTCCACACCAGATAATGTTTCTTGGAGACATCCCACAATGGGCTCTGTTTTTATTGGAAGACTCATTGAACATATGCAAGAATATGCCTGTTCCTGTGATGTGGAGGAAATTTTCCGCAAGGTTCGATTTTCATTTGAGCAGCCAGATGGTAGAGCGCAGATGCCCACCACTGAAAGAGTGACTTTGACAAGATGTTTCTACCTCTTCCCAGGACATTAA

GSDMD nucleotide sequence (SEQ ID NO: 2):

ATGGGGTCGGCCTTTGAGCGGGTAGTCCGGAGAGTGGTCCAGGAGCTGGACCATGGTGGGGAGTTCATCCCTGTGACCAGCCTGCAGAGCTCCACTGGCTTCCAGCCCTACTGCCTGGTGGTTAGGAAGCCCTCAAGCTCATGGTTCTGGAAACCCCGTTATAAGTGTGTCAACCTGTCTATCAAGGACATCCTGGAGCCGGATGCCGCGGAACCAGACGTGCAGCGTGGCAGGAGCTTCCACTTCTAC GATGCCATGGATGGGCAGATACAGGGCAGCGTGGAGCTGGCAGCCCCAGGACAGGCAAAGATCGCAGGCGGGGCCGCGGTGTCTGACAGCTCCAGCACCTCAATGAATGTGTACTCGCTGAGTGTGGACCCTAACACCTGGCAGACTCTGCTCCATGAGAGGCACCTGCGGCAGCCAGAACACAAAGTCCTGCAGCAGCTGCGCAGCCGCGGGGACAACGTGTACGTGGTGACTGAGGTGCTGCAGACACAGAAGGAGGTGGAAGTCACGCGCACCCACAAGCGGGAGGGCTCGGGCCGGTTTTCCCTGCCCGGAGCCACGTGCTTGCAGGGTGAGGGCCAGGGCCATCTGAGCCAGAAGAAGACGGTCACCATCCCCTCAGGCAGCACCCTCGCATTCCGGGTGGCCCAGCTGGTTATTGACTCTGACTTGGACGTCCTTCTCTTCCCGGATAAGAAGCAGAGGACCTTCCAGCCACCCGCGACAGGCCACAAGCGTTCCACGAGCGAAGGCGCCTGGCCACAGCTGCCCTCTGGCCTCTCCATGATGAGGTGCCTCCACAACTTCCTGACAGATGGGGTCCCTGCGGAGGGGGCGTTCACTGAAGACTTCCAGGGCCTACGGGCAGAGGTGGAGACCATCTCCAAGGAACTGGAGCTTTTGGACAGAGAGCTGTGCCAGCTGCTGCTGGAGGGCCTGGAGGGGGTGCTGCGGGACCAGCTGGCCCTGCGAGCCTTGGAGGAGGCGCTGGAGCAGGGCCAGAGCCTTGGGCCGGTGGAGCCCCTGGACGGTCCAGCAGGTGCTGTCCTGGAGTGCCTGGTGTTGTCCTCCGGAATGCTGGTGCCGGAACTCGCTATCCCTGTTGTCTACCTGCTGGGGGCACTGACCATGCTGAGTGAAACGCAGCACAAGCTGCTGGCGGAGGCGCTGGAGTCGCAGACCCTGTTGGGGCCGCTCGAGCTGGTGGGCAGCCTCTTGGAGCAGAGTGCCCCGTGGCAGGAGCGCAGCACCATGTCCCTGCCCCCCGGGCTCCTGGGGAACAGCTGGGGCGAAGGAGCACCGGCCTGGGTCTTGCTGGACGAGTGTGGCCTAGAGCTGGGGGAGGACACTCCCCACGTGTGCTGGGAGCCGCAGGCCCAGGGCCGCATGTGTGCACTCTACGCCTCCCTGGCACTGCTATCAGGACTGAGCCAGGAGCCCCACTAAPCA3 nucleotide sequence (SEQ ID NO: 3):

TGTCAACATAGTGTGACGGGAAGGAGCATGATGAGACAGAAGGAAGGTTTAAACTGGGAAATCTGAGAAATGGTATGGTTGTATGTGGGTTGGCATTCTTGCATGATGGGAGTGGCCACCTGCTTTCATATTCTGAAGTCAGAGTGTTCCAGACAGAAGAAATAGCAAGTGCCGAGAAGCTGGCATCAGAAAAACAGAG GGGAGATTTGTGTGG

PEG3 nucleotide sequence (SEQ ID NO: 4):

GAAAGAGAAAGAGAATGGGACGGCATGTGACTGCCTGATGAAGTTGGCGTGCTTGCTCAAAAGTTCTGCGAGATTGACGGCTCTCTGGATTTGAGCCAAGGACACGCCTGGGAAGCCACGGTGACCTCACAAGGCCCGGAATCTCCGCGAGAATTTCAGTGTTGTTTTCCTCTCTCCACCTTTCTCAGGGACTTCCGAAACTCCGCCTCTCCGGTGACGTCAGCATAGCGCTGCGTCAGACTATAAACTCCCGGGTGATCGTGTTGGCGCAGATTGACTCAGTTCGCAGCTTGTGGAAGATTACATGCGAGACCCCGCGCGACTCCGCATCCCTTTGCCGGGACAGCCTTTGCGACAGCCCGTGAGACATCACGTCCCCGAGCCCCACGCCTGAGGGCGACATGAACGCGCTGGCCTTGAGAGCAATCCGGACCCACGACCGCTTTTGGCAAACCGAACCGGAC

EGFP nucleotide sequence (SEQ ID NO: 5):

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG

2) AAV virus packaging and concentration

A. Will be about 2 x 10 a day in advance ⁷ The 293FT cells were plated onto T175 flasks (25 ml DMEM medium) in 5% CO ₂ Incubating for about 16-24 hours in a 37 ℃ incubator;

B. preparation of PEI-DNA mixture: preparing a 1.5ml centrifuge tube and a 15ml centrifuge tube, uniformly mixing 10.5ug of AAV virus core plasmid, 10.5ug of capsid plasmid, 30ug of Helper plasmid and 3ml of Opti-MEM in the 50ml centrifuge tube, uniformly mixing 150ul PEI (100 uM concentration) and 3ml of Opti-MEM in the 15ml centrifuge tube, incubating for 5 minutes at room temperature, fully mixing the two, and incubating for 20 minutes at room temperature;

C. carefully sucking the supernatant of the 293T cells, carefully cleaning the supernatant once by using 3ml of DPBS, sucking the DPBS, adding 18ml of Opti-MEM culture medium into 50ml of a centrifuge tube in the step 2, repeatedly blowing and beating for a plurality of times, and then adding the mixture into a 293FT cell culture bottle;

D. the dish in step 3 was placed in a container containing 5% CO ₂ After about 6 hours incubation at 37℃in an incubator, the whole supernatant was aspirated and 25ml of complete DMEM medium was added;

E. collecting the supernatant respectively at 48 hr and 72 hr, mixing, and filtering with 0.45um filter membrane;

F. adding 12.5mL 5 XPEG 8000 precipitated virus, mixing completely, and standing overnight at 4deg.C;

G. centrifuging at 4000g for 30min at 4 ℃ on the next day;

H. the supernatant was discarded and 500uL PBS was added to resuspend virus;

I. viral titers were determined using qPCR.

3) AAV infection of target cells

A. The day before infection C4-2, rwpe-1, FB cells were plated into 6-well plates, 6X 10 per well ⁵ (C4-2、RWPE-1)、3×10 ⁵ (FB) to 6-well plate;

B. various viruses were added at MOI 1000 on the day of infection;

C. culturing in a cell culture box.

4) Promoter start detection

Cells expressing green fluorescent protein were observed by confocal microscopy.

3. Experimental results

After AAV viruses carrying different promoters infect different cells, the results show that constitutive promoter SFFV can normally promote the expression of the downstream EGFP gene in three cells (FIG. 1, FIG. 2 and FIG. 3), while tumor specific promoters PCA3 and PEG3 show poor promoter capacity in normal cells (FIG. 1 and FIG. 2), but can effectively promote the expression of EGFP in prostate cancer cells (FIG. 3). At the same time, the results show that CEA specific promoter has a small leakage initiation in the normal prostate cell line RWPE-1 (FIG. 2).

Example 2 in vitro verification of the killing Capacity of specific promoter-initiated killer Gene against tumor cells

1. Experimental materials

(1) Prostate cancer cell C4-2, normal prostate cell Rwpe-1; (2) live Tox Red dead cell indicator reagent.

2. Experimental method

(1) C4-2, RWPE-1 cells were plated at 4X 10 the day before cell transfection ⁵ Well inoculated into 12-well plates;

(2) the culture medium is discarded from each hole on the day of cell transfection, 750ul of complete culture medium is replaced, and the cells are placed in a cell culture box for culturing for 30min;

(3) diluting 0.75ug plasmid with 38uL serum-free DMEM medium, diluting 2.25uL Ployjet reagent with 38uL serum-free DMEM medium, immediately adding diluted Polyjet into diluted solution, mixing, and standing at room temperature for 15min;

(4) adding the mixed solution obtained in the step (3) into a corresponding hole, adding a 1 XC.LIVE Tox Red dead cell indication reagent, uniformly mixing the mixed solution with the Chinese character '8', adding the mixed solution into the corresponding hole, immediately placing the mixed solution into CellCyte to collect fluorescent signals (indicating dead cells), collecting 4 fields of view in each hole, and collecting the fluorescent signals once every 3 hours;

(5) after the collection is completed, the fluorescence areas of all the holes are separated, and the death condition of the cells is counted.

3. Experimental results

The structure shows that cells transfected with AAV-SFFV-Casp1act-GSDMD and AAV-PCA3-Casp1act-PEG3-GSDMD showed massive death in C4-2 cells compared to control AAV empty vector (FIG. 4); in the prostate normal cell RWPE-1, only the plasmid AAV-SFFV-Casp1act-GSDMD transfected with the constitutive promoter is dead, and no obvious death phenomenon occurs in the cells after AAV empty vector transfection and specific promoter AAV-PCA3-Casp1act-PEG3-GSDMD transfection (FIG. 5). The double-specific promoter can improve the safety on the premise of ensuring the effectiveness by controlling the expression of the killer genes.

Example 3 in vitro verification of the efficacy and safety of specific promoter-initiated killer genes for tumor cell killing

1. Experimental materials

(1) Nude mice; (2) prostate cancer cells C4-2.

2. Experimental method

A. Subcutaneous tumor formation in mice

(1) Digesting C4-2 tumor cells, and adjusting cell concentration to 2×10 ⁷ Individual cells/mL

(2) Selecting a right armpit needle of a mouse, passing forward, slightly moving the needle slightly before injection, and ensuring the needle is positioned under the skin;

(3) after the whole needle is slowly inserted into the skin, 100 mu L of cell suspension is slowly injected, and obvious bulge can be seen;

(4) after the injection is finished, slowly rotating to withdraw the needle head, so as to avoid liquid leakage as much as possible;

(5) tumor volume measurement is performed after subcutaneous tumor formation, and tumor volume calculation is performed according to the formula:

volume = long diameter x short diameter x 1/2

Mice were divided into control (Con) and experimental (AAV) groups according to tumor volume;

B. mice dosing and tumor volume and body weight monitoring

(1) 100uL A by tail vein injectionAV virus (1×10) ¹⁰ Experimental group), control group was injected with 100uL PBS;

(2) tumor volume, mouse body weight were measured and recorded every 3 to 4 days;

(3) the biochemical detection of the mouse blood is carried out by each blood biochemical analyzer.

C. Biochemical detection of mouse blood and organ coefficients

(1) After 4 weeks of administration, the eyesockets of all mice are subjected to blood sampling, serum is collected, and the contents of glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, alkaline phosphatase and transglutaminase in the blood of the mice are detected by a blood biochemical analyzer;

(2) after completion of blood collection, the mice were euthanized, and the main organs (heart, liver, spleen, lung, kidney, brain) were collected and organ coefficients were calculated.

3. Experimental results

The results of the mice experiments showed that AAV-treated groups can significantly inhibit tumor growth (fig. 6), and that the mice had no significant differences in body weight from the control group (fig. 7). The double-promoter start killing gene AAV virus can effectively inhibit the growth of tumor and has no obvious toxic or side effect.

Compared with the control group, AAV-PCA3-CASP1-PEG3-GSDMD has no obvious difference in the content of glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, alkaline phosphatase and glutaminyl transferase in blood (figure 8), and also has no obvious difference in the organ coefficients of heart, liver, spleen, lung, kidney and brain (Table 1). The 5AAV-PCA3-CASP1-PEG3-GSDMD was shown to have no significant toxicity to mice.

TABLE 1 organ coefficients of major organs

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. A vector of a bispecific promoter, characterized in that the bispecific promoter is two specific promoters;

the carrier comprises: the double-specificity promoters are respectively connected with target genes;

the gene of interest comprises at least one of the following combinations: a combination of Casp1act and GSDMD, a combination of GSDMD C and GSDMD N;

the two components in each combination of the target genes can be respectively called a target gene 1 and a target gene 2;

preferably, the two promoters of the bispecific promoter may be called specific promoter 1, specific promoter 2, respectively;

preferably, the structure of the vector of the bispecific promoter comprises a specific promoter 1, a target gene 1, a specific promoter 2 and a target gene 2 which are connected in sequence;

preferably, the specific promoter is a cancer-associated specific promoter;

preferably, the cancer comprises squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, scrotum cancer, esophageal cancer, biliary tract tumors, and cancers of the head and neck;

preferably, the two promoters corresponding to the bispecific promoter are any two of PCA3, PEG3, human telomerase reverse transcriptase promoter, survivin promoter, medkine promoter, carcinoembryonic antigen promoter, MUC-1 promoter and probasin gene promoter;

preferably, the vector comprises a plasmid, lentiviral vector, retroviral vector, adenoviral vector, adeno-associated viral vector, linear polynucleotide;

preferably, the vector of the bispecific promoter comprises a vector with the structure AAV-PCA3-Casp1act-PEG 3-GSDMD;

preferably, the vector of the bispecific promoter comprises a Casp1act nucleotide sequence as shown in SEQ ID No. 1;

preferably, the vector of the bispecific promoter comprises a GSDMD nucleotide sequence as shown in SEQ ID NO. 2;

preferably, the vector of the bispecific promoter comprises the PCA3 nucleotide sequence as shown in SEQ ID NO. 3;

preferably, the vector of the bispecific promoter comprises the PEG3 nucleotide sequence as shown in SEQ ID NO. 4;

preferably, the vector structure of the bispecific promoter comprises the nucleotide sequences of SEQ ID NO. 3, SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 2, which are linked in sequence.

2. A method of constructing a vector for a bispecific promoter, wherein the method of constructing is for constructing the vector of claim 1, the method of constructing comprising:

obtaining a bispecific promoter;

assembling the combination of the target genes and the bispecific promoter on a vector to obtain the vector;

preferably, the two promoters of the bispecific promoter comprise any two of PCA3, PEG3, human telomerase reverse transcriptase promoter, survivin promoter, midkine promoter, carcinoembryonic antigen promoter, MUC-1 promoter, probasin gene promoter.

3. The method of claim 2, wherein the vector comprises a plasmid, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a linear polynucleotide;

preferably, the vector is an AAV viral core plasmid.

4. A method of construction according to claim 3, characterized in that the method of construction comprises any one of the following:

a. amplifying the combination of the target genes, amplifying a bispecific promoter, connecting the target genes with the specific promoter, and cloning the connector onto the vector to obtain the vector comprising the bispecific promoter and the target genes;

b. amplifying the target gene and the specific promoter, and then directly cloning the amplified target gene and the specific promoter onto the vector to obtain the vector comprising the bispecific promoter and the target gene.

5. A host cell comprising the vector of claim 1;

preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell;

preferably, the prokaryotic cell comprises bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, rickettsia;

preferably, the eukaryotic cells include mammalian cells, insect cells, plant cells, yeast cells;

preferably, the mammalian cells include fibroblasts, 293T cells.

6. A virus comprising the vector of claim 1;

preferably, the virus comprises a lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, papovavirus, baculovirus, papillomavirus;

preferably, the virus is an adeno-associated virus.

7. A method of producing the virus of claim 6, comprising the steps of:

transfecting the vector of claim 1 into a host cell; and

amplifying and expressing the transfected host cells to obtain the virus;

preferably, the virus is an adeno-associated virus;

preferably, the host cell comprises a prokaryotic cell, a eukaryotic cell;

preferably, the mammalian cells include fibroblasts, 293T cells.

8. A pharmaceutical composition comprising the vector of claim 1, the host cell of claim 5, the virus of claim 6;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable auxiliary ingredient;

preferably, the adjunct ingredients include solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, gums.

9. Use of the vector of claim 1, the construction method of any one of claims 2-4 or the host cell of claim 5 in the preparation of a virus or plasmid;

preferably, the virus is an adeno-associated virus.

10. Use of the vector of claim 1, the construction method of any one of claims 2-4, the host cell of claim 5, the virus of claim 6, or the preparation method of claim 7 for the preparation of a medicament for treating a tumor;

preferably, the cancer comprises squamous cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, adenocarcinoma of the lung, squamous carcinoma of the lung, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal carcinoma, pancreatic carcinoma, glioblastoma, cervical carcinoma, ovarian carcinoma, liver carcinoma, bladder carcinoma, liver carcinoma, colon carcinoma, rectal carcinoma, colorectal carcinoma, endometrial or uterine carcinoma, salivary gland carcinoma, renal carcinoma, prostate carcinoma, vulval carcinoma, thyroid carcinoma, hepatic carcinoma, anal carcinoma, penile carcinoma, scrotum carcinoma, esophageal carcinoma, biliary tract tumor, and head and neck carcinoma.