WO2002056917A1 - Virus recombinant capable d'eradiquer de maniere specifique une tumeur associee au virus eb et son procede de construction - Google Patents

Virus recombinant capable d'eradiquer de maniere specifique une tumeur associee au virus eb et son procede de construction Download PDF

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WO2002056917A1
WO2002056917A1 PCT/CN2002/000025 CN0200025W WO02056917A1 WO 2002056917 A1 WO2002056917 A1 WO 2002056917A1 CN 0200025 W CN0200025 W CN 0200025W WO 02056917 A1 WO02056917 A1 WO 02056917A1
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virus
gene
adenovirus
cells
promoter
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PCT/CN2002/000025
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Chinese (zh)
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Qijun Qian
Xiaoyan Che
Shuntang Sham
Mengchao Wu
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Virgene Biotechnology Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of life sciences, and in particular, to a tumor cell capable of proliferation, expression of late genes, and high expression of anti-cancer genes in tumor cells infected or latently infected by Epstein-Barr virus, while not proliferating in Epstein-Barr virus-negative normal cells and Recombinant cytolytic virus that does not express late genes, and methods for its construction and proliferation.
  • Epstein-Barr virus (called the EB virus according to the conventional cylinder, recorded as EBV, the same below) can be latently infected in all cells of Barkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, and its viral gene EBNA-1 is found in the above tumor cells. expression, and are very low in normal cells, in peripheral blood mononuclear cells positive rate of 10-5 -10_ 6, in other normal adult cells almost were negative.
  • HPV virus can be latently infected in 90% of cervical cancer cells.
  • the cervical cancer cells usually express the HPV virus E6 and E7 genes, and the expression rate of this gene in normal cells is extremely low or negative. Therefore, in tumor patients associated with this virus, the viral protein can be a very good tumor marker.
  • the viral proteins on these tumor cells have been used as specific targets for immunotherapy.
  • tumor cells develop There are many changes, such as the down-regulation of MHC class 1 gene expression and antigen presentation in many tumor cells, and the lack of a second signal of immune cell stimulation, so it cannot effectively activate cytotoxic T cells to kill tumor cells. Therefore, clinical immunotherapy is effective And very unsatisfactory.
  • Gene therapy is a new method for treating malignant tumors that has emerged in recent years.
  • the gene transfection method is divided into two methods: viral method and non-viral method.
  • Viral methods usually use retrovirus, adenovirus, adeno-associated virus, herpes simplex virus and vaccinia virus.
  • Retroviruses have a high transfection rate in vitro, but have a low virus titer, a low transfection rate in vivo, and can only infect cells in the dividing stage. They also integrate into the chromosome and have the potential to become cancerous.
  • Adeno-associated virus has the ability to transfect dividing and resting cells, and can be expressed persistently.
  • Non-viral methods include methods such as liposomes and gene guns, which have a shorter transfection gene expression time and a lower transfection rate.
  • Adenovirus is currently the most common viral vector in tumor gene therapy. It has been widely used in human gene therapy programs. It has the characteristics of easy production and purification. It can effectively transfect dividing and resting cells in vivo and in vitro, without carcinogenicity. .
  • the main obstacle of tumor gene therapy is that it cannot effectively transfect the target gene specifically to all tumor cells, and the efficacy of gene therapy is closely related to the number of gene transfection of tumor cells. Therefore, research and development of specific transfection of all Viral vector systems of tumor cells are essential in tumor gene therapy.
  • the modified virus can only specifically proliferate and lyse tumor cells in tumor cells, and then The virus particles are released, re-infect other tumor cells, and proliferate and lyse again. This has an amplification effect. Because the virus can spread to various tissues and organs throughout the body, it can infect all tumor cells, thereby killing local and metastatic tumors, and Does not affect normal cells.
  • the first is the adenovirus ONYX-015 with E1B 55Kda protein inactivation.
  • the main mechanism is: Many researchers have found that P53 plays a large role in the process of canceration. P53 mutation or inactivation can make DNA Damaged cells continue to divide and replicate, and cancer can occur when other genes are mutated. P53 is also the main antiviral protein of the host cell. P53 can be activated after the virus infects the cells in normal cells. For example, the E1A region of the gene necessary for adenovirus proliferation can cause abnormal cell proliferation and activate P53 at the same time, eventually leading to cell apoptosis and Virus replication Termination.
  • the adenovirus When the adenovirus lacks the 55Kda protein in the E1B region, because P53 has normal functions in normal cells, it is quickly activated, which causes cell apoptosis, prevents the adenovirus from effectively replicating and proliferating, and terminates the infection. In tumor cells with P53 mutations or inactivation, P53 cannot be activated after the virus infection, so the adenovirus lacking E1B can still replicate and proliferate, causing the virus to replicate in tumor cells in large numbers, eventually leading to tumor cell lysis and death, and New viruses are released to infect other tumor cells, which infects all tumor cells and dies. ONYX-015 has significant effects on tumors with P53 mutations and dysfunction in vivo and in vitro.
  • herpes simplex virus Significant neurotrophic characteristics. In resting cells, its ribonucleotide reductase and thymidine deoxynucleotide kinase (HSV-TK) are genes necessary for viral DNA replication. This is found in fast-growing cells such as tumor cell viruses. DNA replication does not require these two genes. This is because sufficient numbers of nucleotides are present in fast-growing cells. Therefore, the virus can also be replicated in the absence of HSV-T. Because normal central nervous cells are at rest Intracranial tumor cells are in the growth phase. Therefore, Martuza RL et al.
  • the third example is from Rodriguez R and other reports.
  • the prostate characteristic antigen (PSA) promoter was used to insert the upstream region of the adenovirus type 5 essential gene (E1A), named CN706.
  • E1A adenovirus type 5 essential gene
  • the expression of the E1A gene of the virus is strictly regulated by the PSA promoter.
  • Control In LNCaP, a prostate cancer cell line that secretes PSA, the expression of E1A gene is reduced by 99%, and its adenovirus proliferation is significantly reduced.
  • the virus titer is 150 times lower than that of LNCaP, a prostate cancer cell line that secretes PSA.
  • An object of the present invention is to provide a type of proliferative recombinant virus capable of specifically killing EB virus-related tumors and a method for constructing the same, that is, the virus can selectively replicate and proliferate in EB virus infected or latently infected tumor cells, and In EB virus-negative normal cells, there is almost no replication and no proliferation, thereby specifically inhibiting or killing EB virus infected or latently infected tumor cells.
  • the present invention provides a proliferative recombinant virus capable of specifically killing EB virus-related tumors, and has a target gene inserted into a non-proliferative essential region of the virus genome.
  • the target gene is one of (1) an tumor suppressor gene, (2) a vascular suppressor gene, (3) a cytokine gene, (4) a prodrug converting enzyme gene, and (4) an apoptosis gene.
  • the target gene carried by the recombinant virus provided by the present invention is a tumor suppressor gene, which suppresses tumor cell growth.
  • the tumor suppressor gene is selected from the group consisting of: P53, Rb, leg, VHL, and APC.
  • the target gene carried by the above-mentioned recombinant virus provided by the present invention is a vascular inhibitory gene.
  • the vascular suppressor gene suppresses tumor neovascularization and can block the nutritional supply of tumor cells, thereby causing the tumor cells to suffer from nutritional deficiencies. Upon death, the tumor shrank significantly or even completely subsided. At the same time, the formation of tumor neovascularization is inhibited, which also achieves the purpose of blocking the pathway of tumor metastasis.
  • Angiosuppressor genes are selected from the group consisting of endostatin gene, angiostatin gene, Kringlel-4 structure, Kringlel-5 structure, Kringlel-3 structure, Kringlel-3 plus Kringle5 structure, and interferon in plasma plasminogen - ⁇ gene, interferon-P gene, interferon- ⁇ gene, thrombospondin gene, platelet factor 4 gene, plasminogen activating factor inhibitor (PAC) gene, interleukin-12, and fibronectin gene.
  • Angiosuppression gene contains a secretory signal encoding
  • the secretory signal peptide can be selected from the group consisting of: angiogenesis inhibitor signal peptide, M-oncogene protein signal peptide, immunoglobulin K chain signal
  • the purpose of carrying the foregoing recombinant virus provided by the present invention is Gene
  • the cytokine gene has the functions of activating immune cells and increasing hematopoietic function.
  • the cytokine gene is selected from the group consisting of: interleukin 2, interleukin 12, granulocyte-single colony stimulating factor, tumor necrosis factor, interferon- ⁇ , interferon ⁇ , interferon- ⁇ , Light and Flt3 ligands.
  • the target gene carried by the above-mentioned recombinant virus provided by the present invention is a prodrug converting enzyme gene.
  • the prodrug converting enzyme gene can transform a non-toxic drug into a toxic drug, thereby enhancing the killing of tumor cells.
  • the prodrug converting enzyme gene is selected from the group consisting of herpes simplex virus thymidine kinase, varicella-zoster virus thymidine and E. coli cytosine deaminase.
  • the gene of interest carried by the above-mentioned recombinant virus provided by the present invention is an apoptotic gene.
  • the apoptotic gene can cause eukaryotic cell apoptosis.
  • the apoptotic gene includes one of the following genes: ICE, capase -3, capase-8, capase-9.
  • trans-acting factors such as transcription factors
  • cis-acting elements When certain transcription factors are lacking or present, it can affect the transcription level of genes.
  • the virus When the host cell is infected with a virus or a latent infection, the virus can change the transcriptional regulatory factors in the cell, which is conducive to the expression of the virus's genes, the replication and proliferation of the virus.
  • certain cis-acting elements of a viral gene are used to control a target gene, so that the target gene is specifically expressed in the virus-infected or latently infected cells, but not expressed or expressed at low levels in virus-negative cells.
  • This modified virus vector can be used to kill specific target cells infected or latently infected by a certain virus in certain cell mixtures. By administering the modified virus, it can selectively proliferate in such target cells.
  • the target cells are selectively killed by the proliferating virus; in vitro culture or in animals by mixing the modified virus with the cell complex, the virus can only proliferate in the target cell, that is to say, except for the target cell, Other cells do not Can be killed by this virus. Because the virus multiplies and expands in the target cell, the target cell in the mixed cell is killed. Once the target cell is destroyed, the virus can no longer reproduce.
  • the cell-specific response element is composed of a cis-acting element that is specifically activated by EB virus-infected or latently infected cells.
  • the cis-acting element EB virus Orip combined with a Bam HI C-promoter controlled reporter gene.
  • the expression level of latently infected cells (EBNA-1 positive) cells is about 1000 times higher than that of EB virus negative (EBNA-1 negative) cells.
  • EBNA-1 positive latently infected cells
  • EBNA-1 negative EB virus negative
  • the reporter gene controlled by the promoter (mini-HSV-TK) or the basic promoter of SV40 (mini-SV40 promoter) is more expressed in EB virus latently infected cells (EBNA-1 positive) cells than in EB virus negative (EBNA- 1 negative) 100-1000 times higher expression.
  • a proliferative recombinant virus capable of specifically killing EB virus-associated tumor cells is proposed in the present invention, and at least one of the genes necessary for virus proliferation is specifically infected with EB virus or latently infected cells.
  • sexually activated cis-acting element control It contains specific cis-acting elements inserted in the region between the transcription start site and the coding start site of the gene necessary for virus proliferation.
  • the cis-acting element is specifically activated in EB virus-infected or latently infected cells to produce transcriptional activity, while it is not activated in EB-virus-negative cells to produce transcriptional activity.
  • the cis-generating element is selected from: Orip in EB virus, family of 30bp repeats in EB virus (denoted as FR), EB virus Bam HI C-promoter, Orip in EB virus combined with EB virus Bam HI C- Promoter, FR in EB virus Orip combined with herpes simplex virus thymidine kinase basic promoter (mini-HSV-TK) or SV40 basic promoter (mini-SV40 promoter), and in EB virus infected or latently infected cells Specific activated cis-acting element. What is necessary for the above virus to multiply is an early-expressing gene of the virus, such as the herpes simplex virus early-epitope ICE4.
  • the virus may be an adenovirus.
  • the genes necessary for virus proliferation contain at least one of the following early expressed genes of adenoviruses: E1A, E1B, E2, E4.
  • the proliferative recombinant virus specifically killing EB virus-related tumor cells can be used with chemotherapeutic drugs (such as cisplatin, 5-fluorouracil, mitomycin C, carbon town, cyclophosphamide, etc.), biological Toxins (such as snake venom) and monoclonal antibodies (such as anti-nasopharyngeal cancer cell antibodies) can be combined to form a pharmaceutical composition, which is more effective as an anti-tumor drug.
  • chemotherapeutic drugs such as cisplatin, 5-fluorouracil, mitomycin C, carbon town, cyclophosphamide, etc.
  • biological Toxins such as snake venom
  • monoclonal antibodies such as anti-nasopharyngeal cancer cell antibodies
  • Two vectors containing the left arm and the right arm of the adenovirus are co-transfected into cells that will produce the recombinant adenovirus.
  • At least one of the two adenoviral vectors has an adenoviral non-essential region inserted into the gene of interest, and at least one of the two adenoviral vectors has an adenoviral early expression gene E1A, E1B, E2 and E4 in the vector.
  • a certain cis-acting element is inserted in the region between the point and the coding start site, and the cis-acting element is specifically activated in EB virus-infected or latently infected cells to produce transcriptional activity, but not in EB-virus-negative cells.
  • the cis-acting element contains at least one of the following sequences: Orip in EB virus, FR in EB virus, EB virus Bam HI C-promoter, Orip in EB virus combined with EB virus Bam HI C-promoter, EB FR in the virus Orip combined with herpes simplex virus thymidine stimulated basic promoter (recorded as mini-HSV-TK) or SV40 basic promoter (recorded as mini-SV40 promoter), and in EB virus infection or latent infection Specific activated cis-acting elements in cells.
  • Inserting specifically activated cis-acting elements in EB virus-infected or latently-infected cells into the region between the transcriptional start site and the coding start site of the early adenovirus gene can be accomplished by the following methods: ) Technology to make an obscure site in the region between the transcriptional start site and the coding start site of the early gene of the adenovirus, and insert the above-mentioned cis-acting element into the site by enzyme digestion, so that the early gene of the adenovirus is affected.
  • Human adenovirus has six different subgenus, which are divided into A, B, C, D, £ and their affinity for host cells, tumorigenicity and disease history are not the same.
  • the present invention further exemplifies the present invention by taking type 5 (Ad5) in the subgenus C of adenovirus as an example, but it is not limited to this example.
  • Adenovirus genes are divided into two categories, early genes and late genes. Early genes include E1A, E1B, E2, and E4. Ela gene is expressed immediately after virus infection (0-2 hours). It is the earliest expressed adenoviral protein. It acts as a transactivation transcription regulator and is necessary for the expression of other early genes and proteins. At the same time, it transactivates the activation of other viruses. child. Therefore, without the expression of the E1A gene, the gene product necessary for adenovirus DNA replication cannot be produced, and the adenovirus cannot effectively replicate and proliferate.
  • Elb gene transcription is activated by the E1A protein, which functions as a late viral gene Necessary for the mRNA to be transported from the nucleus to the cytoplasm. Loss of E1B gene expression can lead to poor gene expression in the later stages of the virus and failure to shut down host cell protein synthesis.
  • the E4 gene is located at the right end of the viral genome, and its open reading frame 3 (ORF3) and ORF6 can increase the mRNA level of the major adenovirus late genes. Lack of ORF3 and ORF6 protein function, its viral titer of 106 times lower than the wild titer.
  • the adenovirus system is composed of two vectors, one vector provides the left arm portion of the adenovirus, and the other vector provides the right arm. These two vectors have at least 500 nt homologous recombination regions, but the transfected cells produce recombinant adenovirus.
  • Vector system plasmid pXCl (Mckinnon, Gene 1982, 19: 39-42), containing the left arm of wild-type Ad5.
  • pBHGlO provides Ad5 right arm lacking E3 region or pBHGE3 provides Ad5 right arm (including E3 region).
  • the transcription start site and coding start site of Ad5 E1A are located at about 560nt and 610nt of the viral genome, respectively. Some viral cis-acting elements are inserted in this region, such as the Orip-BamH IC promoter of Epstein-Barr virus.
  • PCR polymerase chain reaction
  • a similar scheme was also used to insert the viral cis-acting element to regulate the E1B promoter of Elb Ad5. It consists of a single high affinity for Spl as an identifier and a TATAbox. This region is located from 1636 to Sub -1701nt by inserting viral cis The type of action element in this region will provide E1B with cell-specific transcription.
  • the invention also proposes a method for proliferative recombinant virus proliferation, that is, a recombinant virus (such as a recombinant adenovirus) infects mammalian cells infected with EB virus or latently infected, so that the recombinant virus is specifically infected with EB virus or latently infected. Mammalian cells proliferate.
  • a recombinant virus such as a recombinant adenovirus
  • Adenoviruses that can only proliferate in special target cells infected with EB virus or latent infection are used to allow adenoviruses to multiply and cause cell death in the target cells.
  • adenovirus In vitro culture or in animals by mixing modified adenovirus with cell complexes, adenovirus can only proliferate in cells infected with EB virus or latent infection, that is to say, except for cells infected with EB virus or latent infection, Its Other cells cannot be killed by this adenovirus. Because adenovirus multiplies and expands in EB virus infected or latently infected cells, the EB virus infected or latently infected cells in mixed cells are killed. Epstein-Barr virus-infected or latently infected cells are destroyed, and adenoviruses can no longer reproduce.
  • the invention proposes the practical application of the above-mentioned recombinant virus.
  • the recombinant virus (such as recombinant adenovirus) is used to infect EB virus infected or latently infected tumor cells in vitro, resulting in cytotoxicity.
  • Recombinant viruses (such as recombinant adenoviruses) are used to inhibit the growth of tumor cells infected with EB virus or latent infection.
  • Recombinant viruses (such as recombinant adenoviruses) are used in vivo to selectively kill EB virus infected or latently infected tumor cells.
  • the present invention provides a class of proliferative recombinant virus for treating EB virus-related tumors. Animal experiments have proven that this recombinant virus can be used to treat EB virus-related tumors, including nasopharyngeal carcinoma, Hodgkin's lymphoma, and Burkitt's lymphoma. Gastric cancer.
  • the present invention provides a method for constructing a proliferative recombinant virus for treating EB virus-related tumors. This method is easy to implement and can be used to construct a variety of proliferative recombinant viruses for treating EB virus-related tumors.
  • the present invention provides a method for killing EB virus latent infection or infecting tumor cells in vivo and in vitro without affecting EB virus negative normal cells.
  • Example 1 Construction of a vector containing an EB virus cis-acting element
  • pCAT-Basic In the basis vector pCAT-Basic, inserted 3 0bp EB virus family of repeats in Orip at its multiple cloning site (FR, located EB virus 73 3 7 _ 8 l 9 0bp ) combined SV40 basal promoter (mini-SV40 ), pCAT-Basic was purchased from Promega. The 30bp repeat family and mini-SV40 promoter in EB virus Orip were amplified by PCR technology. The former template EB virus DNA was extracted from lymphoma cell line B95-8. For the method for extracting viral DNA, refer to the operating instructions of QIAamp Blood Kit of QIAGEN. The latter template is pCAT-Control (purchased from Promega).
  • primers for FR in Epstein-Barr virus Orip are: Primer 1: 5,-Primer (containing Hind III and Age I restriction sites) GGG AAG CTT ACC GGT GCA TGC AGG AAA AGG ACA AGC (SEQ ID NO.l)
  • Primer 2 3,-Primer (containing part of the mini-SV40 promoter sequence) GAG ATG CAG ATC AAT GGC ACC CCG GGG AAT ACC (SEQ ID NO. 2)
  • the primers for the mini-SV40 promoter are:
  • Primer 3 5,-Primer (containing part of the FR sequence in Orip) GTG CCA TTG ATC TGC ATC TCA ATT AGT CAG (SEQ ID NO. 3)
  • Primer 4 3,-Primer (containing Sal I and Age I restriction sites) GCT AAA GTC GAC ACC GGT AAG CTT TTT GCA AAA GCC TAG (SEQ ID NO.4)
  • the 30bp repeat family (F located in the EB virus 7337-8190bp) was inserted into the multi-cloning site of the Epstein-Barr virus Orip and HSV-TK was basically started.
  • Mini-TK pbluescript II was purchased from Promega.
  • PCR technology was used to amplify the family of 30bp repeats and mini-SV40 promoter in Epstein-Barr virus Orip.
  • the former template EB virus DNA was extracted from the lymphoma cell line B95-8.
  • the latter template is pTAL-Luc (available from Clontech).
  • the primers for FR in Epstein-Barr virus Orip are:
  • Primer 6 3,-Primer (containing part of mini-HSV-TK promoter sequence) AGT CGG GGC GGC AAT GGC ACC CCG GGG AAT ACC (SEQ BO NO.6)
  • the primers for the mini-HSV-TK promoter are Primer 7: 5,-Primer (containing part of the FR sequence in Orip) GGT GCC ATT GCC GCC CCG ACT GCA TCT GC (SEQ ID NO.7)
  • Primer 8 3,-Primer (containing Xba I digestion site) TTT TCT AGA CTT CTG CTT CAT CCC CGT G (SEQ ID NO.8)
  • Fusion sequence of Epstein-Barr virus Orip and mini-TK promoter were used to generate fusion promoter by double overlapping PCR technology (the same method as above), and the fusion promoter PCR fragment was double-digested into pbluescript II KS using Xho I and Xba I (+) Vector (purchased from ATCC, USA) in Xho I and Xba I sites (for the method, please refer to reference 2). This fragment was subjected to DNA sequencing, and its fusion promoter sequence was completely correct, which was recorded as CHEB-FRTK.
  • Example 2 Construction of an attenuated proliferative adenovirus vector that controls the expression of E1A and E1B by an EB virus cis-acting element carrying endostatin or angiostatin.
  • the pXC.l vector was purchased from Mcrobix Biosystem Inc (Toronto), Canada.
  • pXC.1 contains a type 5 adenovirus sequence bp22-5790.
  • a new and unique Age I restriction site was created at 552bp in this vector. This site was located 12bp before the E1A initiation code.
  • the method used was a site-directed mutation double PCR technique (for the method, please refer to the aforementioned reference 1).
  • the primers are
  • Primer 9 5,-Primer (containing EcoR I digestion site) TTC AAG AAT TCT CAT GTT TG (SEQ ID O.9)
  • Primer 10 3,-Primer (Insert an A to generate an Age I restriction site) CAG TCA CCG GTG TCG GAG C (SEQ ID NO.10)
  • Primer 11 5
  • Primer (Insert a T to generate an Age I restriction site) GCT CCG ACA CCG GTG ACT GA (SEQ ID NO.ll)
  • Primer 12 3,-Primer (containing Xba I digestion site) TTC TCT AGA CAC AGG TGA TG (SEQ ID N0.12)
  • the PCR product fragment was inserted into the GEM-T-eas vector (for the method, refer to the operating instructions of Promega Company) and named as pGEM-T-Ela.
  • the fragment was subjected to DNA sequencing, and the sequencing results showed that: T was inserted into the bp552 site of the pXC.l plasmid, thereby generating a new Age I restriction site, and the other sequences were the same as pXC.l.
  • GEM-T-ElA and pXC.1 plasmids were double digested with EcoR I and Xba I to pGEM-T-ElA
  • the mid-cut fragment was inserted into the EcoR I and BamH I sites in the pXC.1 plasmid, and a T was inserted into pXCl 552 to generate an Age I restriction site, which was located 12 bp before the E1A start code.
  • the plasmid was named pXC.l-Age I.
  • CHEB was digested with Age I, and the digested fragments were inserted into the Age I digestion sites in pXC-Age I plasmid.
  • Primers 24 and 19 were used for PCR amplification, and 2050bp were amplified. This indicates that the EB virus Orip
  • the 30bp repeat fragment family combined with the mini-SV40 promoter has been inserted into the Age I site of the XC-Age I plasmid, that is, 12bp upstream of the start codon of the adenovirus type E1A, and named pXC-FRSVElA.
  • the pXC-FRSVEl A vector was double digested with EcoR I and Xba I The 2823bp fragment was recovered and inserted into the EcoBlue I and Xba I restriction sites of pBluescript II SK vector for sequencing. The results confirmed that the 30bp repeat fragment family of the Epstein-Barr virus Orip combined with the mini-SV40 promoter had been inserted into the pXC-Age I plasmid Age I Site.
  • the pXCl vector was purchased from Microbix Biosystem Inc. (Toronto) of Canada.
  • pXCl contains a type 5 adenovirus sequence bp22-5790. Multiple restriction sites were created at 1686bp in this vector, including Bgl II, BamH L Xho I, and Xba I. This restriction site is located between the transcription site of E1B and the start codon. The method uses localized mutation twice. PG technology (see the above reference 1).
  • the primers are
  • Primer 13 5 primer (containing Hind III and Hpa I digestion sites) TTT TGC AAG CTT GTT AAC GCC TTT GTT TGC TGA (SEQ ID N0.13)
  • Primer 14 3,-Primer (Generates four restriction sites) CTC GAG GGA TCC AGA TCT GCG CAT TAT ATA CCC TTT AAG (SEQ ID N0.14)
  • Primer 16 3,-Primer (containing Kpa I digestion site) CCA GAA AAT CCA GCA GGT AAC (SEQ ID NO.16)
  • the CHEB-FRTK was double-digested with Xho I and Xba I, and the fragment was inserted into the Xhol and Xbal digestion sites in pUC-EIB, that is, the E1B transcription start site and the coding start site were forward inserted into the Epstein-Barr virus Orip Family of 30bp repeats combined with the mini-TK promoter, named pUC-ElB-FRTK, using primers 28 and 23 for PCR amplification, which expanded to 1159bp, which indicates that the FR combined mini-TK promoter in Orip virus has been forwarded Insert the UC plasmid Xhol and Xbal sites.
  • pUC-ElB-FRTK Epstein-Barr virus Orip Family of 30bp repeats combined with the mini-TK promoter
  • adeno vector carrying the deletion of the E1 region of the human endostatin gene pCA13 vector was purchased from Microbix Biosystem Inc (Toronto) of Canada.
  • PCA13 contains a type 5 adenovirus sequence bp22-5790 and deletes the El region 342 to The 3523bp fragment was inserted with the human cytomegalovirus (HCMV) IE promoter (-299- +72) and the SV40 poly A tail signal in the E1 deletion region.
  • the human endostatin gene was extracted from fresh normal human liver tissue by using polymerase chain reaction (PCR) technology, and random primers were used as a reverse transcription reaction to amplify the human endostatin gene.
  • PCR polymerase chain reaction
  • Primer 17 GGG GAA TTC ACC ATG GGG GTA CTG CTC ACA CAG AGG ACG CTG CTC AGT CTG GTC CTT GCA CTC (SEQ ID NO.17)
  • Primer 18 CTG CTC AGT CTG GTC CTT GCA CTC CTG TTT CCA AGC ATG GCG AGC CAC CGC GAC TTC CAG (SEQ ID NO.18)
  • Primer 19 GCT CTA GAC TAT TAC TTG GAG GCA GTC ATG AAG CTG TTC TCA ATG CAT AGC ACG ATG TAG GCG TG (SEQ ID NO.19)
  • Primer 18 and primer 19 perform the first PCR amplification to recover a 615bp fragment, and then use primer 17 and primer 19 to perform a second PCR amplification on the 615bp fragment to recover a 647bp fragment.
  • the enzyme was digested with EcoR I + Xba I, and the gene was inserted into the pbluescript IIKS (+) vector (purchased from ATCC, USA) for sequencing. The sequencing results are as follows:
  • PCR polymerase chain reaction
  • total RNA was extracted from fresh normal human liver tissue, random primers were used for reverse transcription reaction, and the first round of PCR was performed, followed by two polymerase chain reactions (PCR) technology (for the method, please refer to reference 1).
  • PCR polymerase chain reactions
  • Primer 20 5'-AGCGAATTCCAAAATGGAACATAAGG-3 '(SEQ ID NO.21)
  • Primer 21 5'-ACACTTTTCCTTGACCTGATTTCAG-3 '(SEQ ID NO.22)
  • Plasma plasminogen 348-343 + 111-94
  • Plasma plasminogen 94-111 + 343-363
  • Primer 23 5'-AGCCTCGAGCTATTACGCTTCTGTTCCTGAG-3 '(SEQ ID O.24)
  • Primer 20 and primer 21, primer 22 and primer 23 were respectively subjected to the first PCR reaction, the reaction products were mixed, and primer 20 and primer 23 were used to perform the PCR reaction again to recover a 1168 bp fragment.
  • EcoR I + Xho I was used to cut the gene, and the gene was inserted into the pbluescript IIKS (+) carrier (purchased from ATCC, USA) for sequencing. The sequencing results are as follows:
  • CA13-human endostatin and pCA13-hmnan angiostatin were digested with Bgl II, and 1237bp (containing human cytomegalovirus (HCMV) IE promoter (-299 + 72)) and human endothelial inhibitory peptides containing M-oncogene protein signal peptide were recovered.
  • Gene and SV40 poly A tail signal) and 758 bp human cytomegalovirus (110 1 ⁇ ) 1 £ promoter (-2 99 — + 72 ), human angiogenesis inhibition and SV40 poly A tail signal
  • Primer 24 GTT AAC GCC TTT GTT TGC TGA (SEQ ID NO.26) Amplified with human endostatin 5 and 3,-primers
  • Primer 25 human endostatin 5 primer (located in M-oncoprotein signal ⁇ human endostatin gene 110-131bp) TCC ACC TGG TTG CGC TCA ACA G (SEQ ID NO.27)
  • primers 24 and 26 can be amplified to 1122bp, indicating that it contains the human cytomegalovirus (HCMV) IE promoter (-299- + 72), the human endostatin gene containing the M-oncogene protein signal peptide, and SV40 poly A
  • the tailing signal is inserted into the pUC-ElB-FRTK Bgl II restriction site in the forward direction and named pUC-ElB-FRTK-endostatinl.
  • primers 24 and 25 can be amplified to 818bp, indicating that it contains human cytomegalovirus (HCMV) IE
  • HCMV human cytomegalovirus
  • the promoter (-299- + 72), the human endostatin gene containing the M-oncoprotein signal peptide and the SV40 poly A tail signal were inserted into the pUC-ElB-FRTK Bgl ll obscure site in the reverse direction. Named pUC-ElB-FRTK -endostatin2.
  • the primers 24 and 28 can amplify to 1364bp, indicating that they contain human cytomegalovirus (HCMV) IE promoter (-299- +72), human angiostatin gene and SV40 poly A tail signal are inserted into ⁇ ⁇ ⁇ in the forward direction.
  • HCMV cytomegalovirus
  • the primer 24 and primer 27 can be amplified to 1440bp, indicating that it contains the human cytomegalovirus (HCMV) IE promoter (-299-+ 72)
  • HCMV human cytomegalovirus
  • the human angiostatin gene and the tailing signal of SV 4 0 poly A were inserted into the pUC-ElB-FRTK Bgl II restriction site, and named pUC-ElB-FRTK -angiostatin2.
  • Hpa I and Kpn I were used to digest pUC-ElB-FRTK-endostatinl, pUC- ⁇ - FRTK-endostatin2, pUC-ElB-FRTK-angiostatinl and pUC-ElB-FRTK- angiostatin2.
  • the recovered fragments were inserted into pXC-FRSVElA respectively.
  • Hpal and Kpn I enzymes Cleavage sites named pXC "RSVElA-FRTKElB-endostatinl, pXC- FRSVElA-FRTKElB-endostatin2, pXC-FRSVElA-FRT ElB- angiostatinl and pXC-FRSVElA-FRTKElB- angiostatm2 0
  • Example 6 endostatin or carrying vessel
  • the statin-producing EB virus cis-acting element controls the recombination of attenuated proliferating adenoviruses expressing E1A and E1B.
  • 293 cell line was purchased from Microbix Biosystem Inc (Toronto) of Canada. It was formed by transforming human embryonic kidney cells with sheared type 5 adenovirus DNA. It contains and expresses type 5 adenovirus E1 region, which is highly transformed by adenovirus DNA. Dyeing rate. A plasmid containing the adenovirus type 5 left arm was combined with a plasmid containing the adenovirus type 5 right arm to co-transfect 293 cells, and infectious adenovirus could be produced by homologous recombination.
  • PBHG10 was purchased from Microbix Biosystems Inc. (Ontario) of Canada. It contains the right arm of adenovirus type 5, but lacks the E3 region.
  • E1A and E1B were activated by the Epstein-Barr virus cis-acting element Orip FR combined with mini-SV40 promoter and Orip FR combined with mini-HSV-TK.
  • EBV FRSVEIA-FRTKElB-endostatinl EBV FRSVEIA-FRTKElB-endostatin2
  • EBV FRSVEIA-FRTKE1B- angiostatinl EBV FRSVEIA-FRTKElB-angiostatin2
  • FRSVEIA-FRTKElB-angiostatin2 respectively CNHKEBV-endostatinl, CNHKEBV-endostatin2, CNHKEBV-angiostatinl and C HKEBV-3Hgiostatin2.
  • EBV FRSVEIA-FRTK ElB-endostatinl (CNHKEBV-endostatinl) is a type 5 adenovirus, which inserts the EB virus cis-acting element Orip FR in conjunction with mini-SV40 to start between E1A and E1B transcription start sites and coding start sites.
  • EBV FRSVEIA-FRTK ElB-endostatin2 (C HKEBV-endostatin2) is an adenovirus type 5, which inserts EB virus cis-acting elements Orip FR in combination with mini-SV40 between E1A and E1B transcription start sites and coding start sites.
  • EBV FRSVEIA-FRTK ElB-angiostatinl (CNH EBV-angiostatinl) is an adenovirus type 5, which is inserted between the transcription initiation sites of E1A and E1B and the encoding initiation site, respectively.
  • the EB virus cis-acting elements Orip FR combined with mini-SV40 promoter and Orip FR combined with mini-HSV-T promoter were added, and after the E1A stop codon, they were newly established between the upstream region of Orip FR combined mini-HSV-TK promoter a Bgl II cleavage site, the cleavage site was inserted into the containing human cytomegalovirus (HCMV) IE promoter (-299 + 72), containing human angiostatin gene and SV40 poly a addition signal
  • HCMV human cytomegalovirus
  • the gene sequence was accompanied by a deletion of 28133-30818bp (partial sequence of the E3 region).
  • the other DNA sequences of the virus were the same as those of the adenovirus type 5.
  • EBV FRSVEIA-FRTK ElB-angiostatin2 (CNHKEBV-angiostatin2) is an adenovirus type 5, which inserts the EB virus cis-acting element Orip FR together with mini-SV40 to start between E1A and E1B transcription start sites and coding start sites.
  • Promoter and Orip FR combined with mini-HSV-TK promoter, and a new Bgl II obscure site was created between the E1A stop codon and the upstream region of Orip FR combined mini-HSV-TK promoter.
  • Example 7 EB virus cis-acting element carrying human endostatin or human angiostatin controls attenuated E1A and E1B expression of attenuated proliferative adenovirus in vitro in EB infected or latently infected tumor cells for proliferation, replication, and high expression Human endostatin or human angiostatin and specifically kills tumor cells.
  • CNHKEBV-endostatinl, CNHKEBV-endostatinl, C HKEBV- angiostatinl and CNHKEBV-angiostatin2 were infected with EB virus-infected lymphoma cell lines Jijoye, 293, and normal human fibroblasts, respectively, and the cells were inoculated into 6-well plates at 2 x 10 5 / well.
  • the DNA extracted from the above CNHKEBV-angiostatinl and CNHKEBV-angiostatin 2 were digested with Bgl ll respectively, electrophoresed with 1% agarose, transferred to nylon membranes, and human angiostatin cDNA fragments were labeled with 32 P (EcoR I Digested CA13-human angiostatin with Xba I, recovered 1168bp) as a probe, performed southern blot hybridization, and used pCA13-human angiostatin as a virus copy number control (for the method, see reference 2), CNHKEBV- angiostatin 1 and C HKEBV- angiostatin 2 in Jijoye and normal human fibroblasts each had a virus copy number of 4 x 10 4 , 4 10 4 and ⁇ 10, ⁇ 10.
  • Example 8 Attenuated human endostatin or human angiostatin EB virus cis-acting element to control E1A and E1B expression of attenuated proliferating adenovirus in SCID mice for treatment of EB virus infection or latently infected tumor cell transplant tumor
  • the pCA14 vector was purchased from Microbix Biosystem Inc (Toronto) of Canada.
  • pCA14 contains the type 5 adenovirus sequence bp22-5790 and deleted the 342 to 3523 bp fragment of the El region.
  • PCR Polymerase chain reaction
  • Primer 29 TTT GTC GAC CAT GGG TCA CCA GCA GTT GGT CAT (SEQ ID N0.31)
  • Primer 30 AGA TCC GCC GCC ACC GCC ACC ACT GCA GGG CAC AGA TGC CCA (SEQ ID NO.32)
  • Primer 31 GGT GGC GGT GGC GGC GGA TCT AGA AAC CTC CCC GTG GCC ACT (SEQ ID NO.33)
  • Primer 32 TAT AAA GTC GAC TCA TTA GGA AGC ATT CAG ATA GCT CG (SEQ ID NO.34)
  • Primer 29 and primer 30, primer 31 and primer 32 were respectively subjected to the first PCR reaction, the reaction products were mixed, and primer 29 and primer 32 were used to perform the PCR reaction again to recover a 1610 bp fragment.
  • This gene was digested with Sal I, and the gene was inserted into pbluescript IIKS (+) vector (purchased from ATCC, USA) for sequencing. The sequencing result was correct.
  • CA14-human IL-12 was partially digested with Bgl II, and 2191bp (containing human cytomegalovirus (HCMV) IE promoter (-299 +72), human IL-1 2 fusion gene, and SV 4 0 poly A tailing) were recovered. Signal), insert it into the Bgl II digestion site in pUC-ElB-FRTK, and apply the PCR technique respectively.
  • Primer 24 GTT AAC GCC TTT GTT TGC TGA (SEQ ID NO.26) was amplified with primer 29 and primer 32 of human interleukin 12, respectively
  • primers 24 and 32 can amplify to 2122bp, indicating that it contains the human cytomegalovirus (HCMV) IE promoter (-299- + 72), the human IL12 fusion gene, and the SV40 poly A tail signal is inserted into pUC-ElB in the forward direction.
  • -FRTK Bgl II restriction site named pUC-ElB-FRTK1-IL12.
  • primers 24 and 29 can amplify to 2122bp, indicating that it contains the human cytomegalovirus (HCMV) IE promoter (-299- + 72), the human IL12 fusion gene and the SV40 poly A tail signal inserted into UC-ElB.
  • -FRTK Bgl II restriction site Named pUC- E1B-FRTK2 -IL12
  • Hpa I and Kpn I were used to cut UC-ElB-FRTKl-IL12 and pUC- ⁇ - FRTK2-IL12.
  • the fragments were recovered and inserted into the Hpal and Kpn I digestion sites in pXC-FRSVElA. They were named pXC-FRSVElA- FRTKElBl-IL12, pXC- FRSVE1A-FRTKE1B2-IL12.
  • Example 11 Recombination of an attenuated proliferating adenovirus that controls the expression of E1A and E1B by an EB virus cis-acting element carrying an interleukin-12 fusion gene
  • 293 cell line was purchased from Microbix Biosystem Inc (Toronto) of Canada. It was transformed from human embryonic kidney cells by shearing type 5 adenovirus DNA. It contains up to type 5 adenovirus E1 region, which is highly transfected with adenovirus DNA. rate. A plasmid containing the adenovirus type 5 left arm was combined with a plasmid containing the adenovirus type 5 right arm to co-transfect 293 cells, and infectious adenovirus could be produced by homologous recombination.
  • PBHG10 was purchased from Microbix Biosystems Inc (Ontario) of Canada. It contains the right arm of adenovirus type 5, but lacks the E3 region. Viral plaques appeared 9-14 days after co-transfection.
  • E1A and E1B were activated by the Epstein-Barr virus cis-acting element Orip FR combined with mini-SV40 promoter and Orip FR combined with mini-HSV-TK.
  • the adenovirus controlled by the daughter, and carrying the human interleukin 12 fusion gene, were named EBV FRSVEIA- FRTKE1B1-IL12 and FRSVEIA-FRTKE1B2-IL12, respectively, and recorded as CNHKEBV1-IL12 and CNHKEBV2-IL12.
  • Ad5 left arm plasmid Ad5 right arm plasmid
  • EBV FRSVEIA-FRTK E1B1-EL12 (CNHKEBV1-IL12) is an adenovirus type 5, which inserts the EB virus cis-acting element Orip FR in conjunction with mini-SV40 to start between the transcription initiation sites and coding initiation sites of E1A and E1B.
  • CNHKEBV2-IL12 is an adenovirus type 5, which inserts the X EB virus cis-acting element Orip FR with mini-SV40 promoter and Orip FR with mini between the E1A and E1B transcription start sites and the coding start site, respectively.
  • -HSV-T promoter, and a new Bgl II restriction site was created between the E1A stop codon and the upstream region of the Orip FR combined mini-HSV-TK promoter.
  • the restriction site contains a human Cytomegalovirus (HCMV) IE promoter (-299- + 72), contains human interleukin 12 fusion gene and SV40 poly A tail signal gene sequence, and is accompanied by deletion of 28133-30818bp (partial sequence of E3 region), virus Other DNA sequences are identical to type 5 adenovirus.
  • HCMV Cytomegalovirus
  • Example 12 EB virus cis-acting element carrying human interleukin 12 fusion gene controls attenuated E1A and E1B expression of attenuated proliferative adenovirus in vitro Proliferation, replication and high expression of human leukocytes in EB infected or latently infected tumor cells And specifically kill tumor cells.
  • CNHKEBV1-IL12 and CNHKEBV2-IL12 were infected with EB virus-infected lymphoma cell lines Jijoye, 293, and normal human fibroblasts, respectively. Cells were inoculated into 6-well plates at 2 x 10 5 / well and infected with recombinant adenovirus C HKEBV1-IL12, respectively. CNHKEBV2-IL12 and wild-type adenovirus type 5 4 X 10 pfu. After 48 hours, the virus titer was measured using a 293 cell line. For specific methods, refer to the aforementioned document 2. The results are shown in Table 4:
  • the DNA extracted from CNHKEBV1-IL12 and CNHKEBV2-IL12 were digested with Bgl II, electrophoresed with 1% agarose, transferred to nylon membrane, and human endostatin cDNA fragments (EcoR I and Xba) were labeled with 32 P, respectively.

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Abstract

L'invention porte sur un virus recombinant reproducteur capable d'éradiquer de manière spécifique des cellules cancéreuses associées au virus EB et son procédé de construction. Un certain élément agissant en cis est inséré dans une région en amont d'un gène prémature du virus de manière que le virus recombinant puisse être reproduit uniquement dans une infection dormante ou dans des cellules infectées du virus EB. Parallèlement, les gènes cibles sont insérés dans une région non reproductrice nécessaire contenue dans un génome viral qui est reproduit dans des cellules cancéreuses de manière que les gènes cibles puissent s'exprimer très efficacement dans les cellules cancéreuses et, par conséquent, éradiquer de manière spécifique l'infection dormante ou les cellules cancéreuses infectées du virus EB. Le virus recombinant peut être utilisé pour traiter les tumeurs associées au virus EB, dont le cancer rhino-pharyngien, la lymphogranulomatose et le cancer gastrique.
PCT/CN2002/000025 2001-01-18 2002-01-17 Virus recombinant capable d'eradiquer de maniere specifique une tumeur associee au virus eb et son procede de construction WO2002056917A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2003047634A3 (fr) * 2001-12-06 2004-01-29 Univ Health Network Acides nucleiques et methodes de traitement de cancers positifs au virus epstein-barr

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CN101497665B (zh) * 2009-02-25 2013-02-06 厦门大学 人纤溶酶原Kringle5多克隆抗体及其制备方法及应用

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WO1995014102A1 (fr) * 1993-11-18 1995-05-26 Rhone-Poulenc Rorer S.A. Adenovirus recombinants codant pour la thymidine kinase lors de therapie genique
WO1995014101A1 (fr) * 1993-11-18 1995-05-26 Rhone-Poulenc Rorer S.A. Adenovirus recombinants pour la therapie genique des cancers
WO1998013383A1 (fr) * 1996-09-24 1998-04-02 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Vecteurs adenoviraux pour mutants de l'interleukine 6 humaine (hil-6) avec action antagoniste sur hil-6, compositions pharmaceutiques a base de ces vecteurs et leurs utilisations
WO2000042208A1 (fr) * 1999-01-14 2000-07-20 The Scripps Research Institute Vecteurs d'adenovirus, lignees cellulaires d'encapsidation, compositions et procedes de preparation et d'utilisation correspondants
CN1261075A (zh) * 1999-11-25 2000-07-26 钱其军 一种能特异性杀灭eb病毒相关肿瘤的重组病毒及其构建方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995014102A1 (fr) * 1993-11-18 1995-05-26 Rhone-Poulenc Rorer S.A. Adenovirus recombinants codant pour la thymidine kinase lors de therapie genique
WO1995014101A1 (fr) * 1993-11-18 1995-05-26 Rhone-Poulenc Rorer S.A. Adenovirus recombinants pour la therapie genique des cancers
WO1998013383A1 (fr) * 1996-09-24 1998-04-02 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti S.P.A. Vecteurs adenoviraux pour mutants de l'interleukine 6 humaine (hil-6) avec action antagoniste sur hil-6, compositions pharmaceutiques a base de ces vecteurs et leurs utilisations
WO2000042208A1 (fr) * 1999-01-14 2000-07-20 The Scripps Research Institute Vecteurs d'adenovirus, lignees cellulaires d'encapsidation, compositions et procedes de preparation et d'utilisation correspondants
CN1261075A (zh) * 1999-11-25 2000-07-26 钱其军 一种能特异性杀灭eb病毒相关肿瘤的重组病毒及其构建方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003047634A3 (fr) * 2001-12-06 2004-01-29 Univ Health Network Acides nucleiques et methodes de traitement de cancers positifs au virus epstein-barr

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