Patent Grant
10039841
The present invention relates to a replicating retrovirus vector system, comprising thymidine kinase (HSV-TK) gene and cytosine deaminase (CD) gene, which enable the gene transfer to tumor tissue for the efficient cancer therapy.
Gene therapy is a general term that indicates a technique to treat disease by replacing an abnormal gene that causes disease in cells or tissues in a patient with a target gene or by inserting a target gene that is helpful for the treatment of disease therein. In the early development days of gene therapy products, the major principle of gene therapy was to induce the specific gene expression by inserting foreign DNA into the target cell chromosome. However, today's gene therapy includes the method using antisense to inhibit the expression of a gene related to a specific disease by using antisense oligodeoxynucleotide or siRNA.
The mentioned gene therapy takes a totally different approach from the conventional treatment methods, so that can investigate a reason of disease at a molecular level for better treatment. This method is also advantageous in reducing unnecessary side effects frequently observed in other treatment methods due to its nucleotide sequence specific function that can eliminate the factors related to major diseases. Such a method targeting gene as the above does not need a separate step of optimization in the course of drug production as long as the nucleotide sequence of a target gene for the control of expression level is identified, indicating the production procedure is simpler than that for an antibody or compound drugs. In addition, this method can target any disease which other methods cannot take it as a target, once a causing gene of the disease is identified, suggesting that gene therapy has a full potential as a next generation treatment agent. Numbers of researches confirmed that the chances of successful treatment for incurable disease, cancer, AIDS, genetic disorder, and neurologic disorder which are hard to treat with the conventional medicinal treatment methods could be increased with gene therapy, and therefore gene therapy is now being applied to actual clinical trials (YOUNG et al, 2006).
A gene medicine is composed of a gene transfer vector and a therapeutic gene. As a tool to deliver a gene in vivo, the gene transfer vector is largely divided into a viral vector and a non-viral vector. The viral vector is prepared by making a virus non-replicable by eliminating most of or a part of essential genes and instead inserting a therapeutic gene therein (Lotze M T et al., Cancer Gene Therapy, 9:692-699, 2002). The viral vector can deliver a gene with high efficiency but has problems of difficulty in mass-production according to the types of virus, causing immune response, toxicity, or introducing replicable virus, etc. The major viral vectors being used for the development of a gene medicine are exemplified by retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and pox virus etc. In the meantime, the non-viral vector does not cause immune response, has low toxicity, and is easy to mass-produce, but is not efficient in delivering a gene and can only induce temporary gene expression.
One of the most frequently used viral vector in the field of clinical trial is a retrovirus vector, which was first used in the first gene therapy clinical trial performed by NIH in 1990. This vector is regarded as the most useful vector for a stable gene insertion. The retrovirus vector based on moloney murine leukemia virus (MoMLV) is widely used in various clinical trials for gene therapy.
The non-replicating retrovirus whose self-replication is defective is suitable for the insertion of a relatively big gene. The titer thereof is 106˜107 pfu/ml, so there is not a big problem in the infection of a target cell with this vector. Also, the packaging cell line has been already established, indicating that the preparation of this vector is easy. In addition, the retrovirus vector can be scaled-up by means of inserting a therapeutic gene in the retrovirus plasmid, with which the packaging cell line is infected to produce the recombinant virus; and infecting a target cell with the produced recombinant virus. However, there is a chance of mutation caused in the course of retroviral integration into chromosome.
Meanwhile, the genome stability of the self-replicable retrovirus vector in the proliferating cell such as a cancer cell has been an issue. Besides, a foreign gene that can be inserted in the self-replicable virus vector for gene therapy is limited up to 1.3 kb in the size, indicating the insertion of various therapeutic genes is not easy (J. of virology, Vol. 75, 6989-6998, 2001).
The therapeutic gene usable for gene therapy is exemplified by the gene inducing cancer cell apoptosis by using a prodrug such as herpes simplex virus thymidine kinase or cytosine deaminase; the cytokine gene accelerating immune response such as interleukin-12 or GM-CSF, etc; and the tumor specific antigen gene such as CEA or Her-2, etc. (Gottesman M M, Cancer Gene Therapy, 10:501-508, 2003). Suicide gene kills cancer cells when it is delivered into the cancer cells. Cytokine gene or tumor specific antigen gene attacks cancer cells by activating immune response against cancer.
Recently, studies have been actively going on about the synthesis technique of enzyme/prodrug that exhibits selective antitumor effect on malignant tumor. Realistically, when suicide gene is expressed in cancer tissues and its precursor is administered in vivo systemically, it does not cause toxicity in normal cells but the precursor is converted into a toxic material to kill tumor cells only in those tumor cells where the therapeutic gene is expressed.
One of the most generally used suicide gene is herpes simplex virus thymidine kinase (HSV-TK). When the prodrug ganciclovir (GCV) which does no harm on cells is converted into a cytotoxic material through enzyme reaction, it acquires bystander effect that can induce the apoptosis of not only the cells harboring the suicide gene but also the neighboring cells by gap junction. The effect and stability of the gene has been confirmed so far until the phase 3 clinical trial (human gene therapy, 4:725-731, 1993; molecular therapy, 1:195-203, 2000).
Another suicide gene is cytosine deaminase (CD), which induces deamination of 5-fluorocytosine (5-FC) to produce a powerful anticancer agent 5-fluorouracil (5-FU). 5-FU is metabolized into 5-fluorouridine triphosphate (5-FUTP) and 5-fluorodeoxyuridine monophosphate (5-FdUMP). 5-FUTP fused to ribonucleic acid interrupts the synthesis of ribosomal ribonucleic acid and messenger ribonucleic acid. In the meantime, 5-FdUMP suppresses thymidine synthase irreversibly, leading to the inhibition of DNA synthesis. Therefore, tumor cells can be selectively killed by converting such prodrugs as GCV and 5-FC into toxic metabolites in the tumor cells expressing TK or CD.
Using more than 2 different therapeutic genes simultaneously for gene therapy is more efficient in disease treatment and particularly in dealing with the disease displaying resistance against a specific gene used for gene therapy. In relation to such a technique, a gene therapy vector system that can express TK and CD simultaneously in the cancer tissue is very much advantageous, particularly for the treatment of such cancers reported to have resistance against TK and CD treatment. However, when HSV-TK and CD are both inserted in a replicating-retrovirus vector (RRV), the genome size becomes 10 kb or more, indicating the insertion of the two genes at the same time in a single retrovirus vector is in fact impossible. When a replicating-retrovirus vector is used for gene therapy, it has to contain a foreign gene in addition to its original endogenous genomic RNA, so that the size of the genomic RNA is getting bigger and has a high potential of therapeutic gene loss because of recombination caused by the additional heterologous nucleotide sequence introduced therein, making the construction of a vector system difficult.
The present inventors tried to develop a safe virus vector system for gene therapy which is free from recombination. As a result, the inventors developed a TK/CD combined self-replicating retrovirus vector system containing both HSV-TK and CD genes with excellent stability but without worry of recombination caused gene loss. The present inventors confirmed that the said vector could induce cancer cell death by using the prodrug GCV or 5-FC and be selectively applied to the treatment of specific cancer that showed resistance against either TK or CD by selecting a proper therapeutic gene and the prodrug thereof. The present inventors further completed this invention by confirming that the said vector thereby could be useful as a pharmaceutical composition for the prevention or treatment of cancer.
It is an object of the present invention to provide a replicating retrovirus vector system harboring thymidine kinase and cytosine deaminase genes for the treatment of cancer.
It is another object of the present invention to provide a recombinant retrovirus containing the retrovirus vector system and a host cell infected with the recombinant retrovirus above.
It is also an object of the present invention to provide a pharmaceutical composition for the treatment of cancer and a composition for gene transfer comprising the recombinant retrovirus above.
It is further an object of the present invention to provide a method for preparing the replicating retrovirus system above.
To achieve the above objects, the present invention provides a replicating retrovirus vector system comprising the first recombinant expression vector containing MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector containing virus Env gene, promoter, and thymidine kinase gene.
In addition, the present invention also provides a replicating retrovirus vector system comprising the first recombinant expression vector containing MuLV Gag-Pol gene, promoter, and thymidine kinase gene; and the second recombinant expression vector containing virus Env gene, promoter, and cytosine deaminase gene.
In addition, the present invention also provides a recombinant retrovirus containing the vector system.
In addition, the present invention also provides a host cell transfected with the recombinant retrovirus above.
In addition, the present invention also provides a pharmaceutical composition for the prevention or treatment of cancer containing the recombinant retrovirus as an active ingredient.
In addition, the present invention also provides a composition for gene transfer for the treatment of cancer comprising the recombinant retrovirus above.
In addition, the present invention also provides a method for preparing the replicating retrovirus vector system comprising the step of preparing the first recombinant expression vector containing MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the step of preparing the second recombinant expression vector containing virus Env gene, promoter, and thymidine kinase gene.
In addition, the present invention also provides a method for preparing the replicating retrovirus vector system comprising the step of preparing the first recombinant expression vector containing MuLV Gag-Pol gene, promoter, and thymidine kinase gene; and the step of preparing the second recombinant expression vector containing Env gene, promoter, and cytosine deaminase gene.
In addition, the present invention also provides a method for treating cancer containing the step of administering the recombinant retrovirus above to a subject in need of the same.
Lastly, the present invention provides a use of the retrovirus above for the production of a drug for the prevention or treatment of cancer.
The TK/CD combined self-replicating retrovirus vector system of the present invention includes both HSV-TK and CD genes; has no worries about losing a therapeutic gene caused by the recombination in the course of virus infection; and has an excellent stability, and also is able to induce cancer cell death by using the prodrug GCV or 5-FC and can apply a therapeutic gene or a prodrug thereof selectively to such cancer that shows resistance against cancer treatment using either TK or CD, so that this system of the invention can be advantageously used as a pharmaceutical composition for the treatment of cancer.
The present invention provides a replicating retrovirus vector system comprising the first recombinant expression vector containing MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector containing virus Env gene, promoter, and thymidine kinase gene.
The thymidine kinase gene herein can be a polynucleotide composed of the nucleotide sequence represented by SEQ. ID. NO: 15. The polynucleotide above is not only the polynucleotide encoding the amino acid sequence of thymidine kinase but also the polynucleotide having the nucleotide sequence which is actually same as the said polynucleotide and a fragment thereof. The polynucleotide which is composed of the same nucleotide sequence as the above can have at least 80%, preferably at least 90%, and more preferably at least 95% homology with the polynucleotide of the invention. As described hereinbefore, the polynucleotide of the invention can have substitution, deletion, or insertion of one or more nucleotides in the sequence, as long as the polynucleotide can encode a protein having the equal activity to the above.
The cytosine deaminase gene above can be optimized by human codon.
The term “optimized by human codon” herein indicates that a codon preferred by a host that designates an amino acid when DNA in the host cell is transcribed and translated is substituted with a human codon so as to increase the expression efficiency of the amino acid or the protein encoded by the nucleic acid.
The cytosine deaminase gene can be the polynucleotide selected from the group consisting of those polynucleotides respectively composed of the sequence represented by SEQ. ID. NO: 16, the sequence represented by SEQ. ID. NO: 17, the sequence represented by SEQ. ID. NO: 18, the sequence represented by SEQ. ID. NO: 19, the sequence represented by SEQ. ID. NO: 20, the sequence represented by SEQ. ID. NO: 21, the sequence represented by SEQ. ID. NO: 22, the sequence represented by SEQ. ID. NO: 23, the sequence represented by SEQ. ID. NO: 24, the sequence represented by SEQ. ID. NO: 25, the sequence represented by SEQ. ID. NO: 26, the sequence represented by SEQ. ID. NO: 27, the sequence represented by SEQ. ID. NO: 28, the sequence represented by SEQ. ID. NO: 29, the sequence represented by SEQ. ID. NO: 30, and the sequence represented by SEQ. ID. NO:31. The polynucleotide can include a variant having the same characteristics as the above.
The thymidine kinase or cytosine deaminase gene can activate a prodrug. The prodrug can be one or more drugs selected from the group consisting of ganciclovir and 5-fluorocytosine. In a preferred embodiment of the present invention, the thymidine kinase gene can activate ganciclovir, and the cytosine deaminase gene can active 5-fluorocytosine.
The said Gag gene can be the polynucleotide encoding kinds of proteins that form the retrovirus core. In the meantime, the Pol gene can be the polynucleotide encoding the retrovirus reverse transcriptase and the Env gene can be the polynucleotide encoding the retrovirus envelope glycoprotein.
The said MuLV-Gag gene is the Gag gene of murine leukemia virus, which can be the polynucleotide composed of the nucleotide sequence represented by SEQ. ID. NO: 32. The said MuLV-Pol gene is the Pol gene of murine leukemia virus, which can be the polynucleotide composed of the nucleotide sequence represented by SEQ. ID. NO: 33. The MuLV Gag-Pol gene can be the polynucleotide composed of the fusion sequence comprising the nucleotide sequence represented by SEQ. ID. NO: 32 and the nucleotide sequence represented by SEQ. ID. NO: 33.
The promoter above can be one of the promoters originated from cancer specific promoters such as MCMV immediate-early promoter, EF1α promoter, HCMV immediate-early promoter, PGK promoter, and hTERT promoter. Herein, the said EF1α promoter can be the polynucleotide composed of the nucleotide sequence represented by SEQ. ID. NO: 34.
The said Env gene can be selected from the group consisting of those Env genes of gibbon ape leukemia virus (GaLV), amphotropic MuLV, xenotropic MuLV, RD114, vesicular stomatitis virus (VSV), and measles virus (MV). The Env gene of gibbon ape leukemia virus can be the polynucleotide composed of the nucleotide sequence represented by SEQ. ID. NO: 35. The said polynucleotide can include a variant having the same characteristics as mentioned above.
In this invention, the term “replicating/replicable” indicates that the virus vector can self-replicate in the cells introduced with the virus genome containing a specific gene or infected with animal cells or the virus vector containing a specific gene.
In this invention, the term “replicating retrovirus vector” indicates the vector that produces non-lytic virus. The vector can specifically infect proliferating cells, that is cancer cells, because it can penetrate into the nucleus when the nuclear envelope is being broken, so that it can prevent an inserted foreign gene from being expressed in other normal cells. Therefore, the vector can deliver a target gene safely into cancer cells and is also replicating to increase the efficiency of gene transfer.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene and GalV-EnV gene in order to express them separately in each vector. The inventors cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors further cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a replicating retrovirus vector system comprising the first recombinant expression vector containing MuLV Gag-Pol gene, promoter, and thymidine kinase gene; and the second recombinant expression vector containing virus Env gene, promoter, and cytosine deaminase gene.
The said vector system has the characteristics explained above. For example, the thymidine kinase gene can activate the prodrug ganciclovir, and the cytosine deaminase gene can active the prodrug 5-fluorocytosine.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them separately in each vector. The inventors cloned P2 promoter and thymidine kinase gene in the vector containing Gag and Pol genes. The inventors further cloned P2 promoter and cytosine deaminase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a recombinant retrovirus containing the vector system above.
The vector system can have the characteristics mentioned above.
However, the recombinant retrovirus above can be produced from the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector comprising virus Env gene, promoter, and thymidine kinase gene. At this time, the first and the second recombinant expression vectors can be included in the recombinant retrovirus independently or together.
In a preferred embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them respectively in each vector, and cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors also cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a host cell transfected with the recombinant retrovirus.
The vector system above can have the characteristics mentioned above.
The host cell herein can be selected from the group consisting of NS/0 myeloma cell, human 293T cell, CHO cell, HeLa cell, CapT cell (human amniotic fluid derived cell), COS cell, canine D17 cell, and feline PG4 cell. In a preferred embodiment of the present invention, the host cell can be human 293T.
The said transfection can be performed by one of the conventional methods well-known to those in the art, which is exemplified by lipofectamine method, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. In a preferred embodiment of the present invention, the transfection was accomplished by lipofectamine method.
The transfected cells were cultured in the conventional medium generally used for animal cell culture. The medium above can be one or more media selected from the group consisting of Eagle's MEM, a-MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, DMEM/F12 combined medium, Way-mouth's MB752/1, McCoy's 5A, and MCDB series media. In a preferred embodiment of the present invention, the medium was DMEM.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them separately in each vector. The inventors cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors further cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a pharmaceutical composition for the prevention or treatment of cancer comprising the recombinant retrovirus above as an active ingredient.
However, the recombinant retrovirus above can be produced from the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector comprising virus Env gene, promoter, and thymidine kinase gene. At this time, the first and the second recombinant expression vectors can be included in the recombinant retrovirus independently or together.
The retrovirus above can target the cell in the course of proliferation which is more precisely cancer cell. The cancer cell herein can include those cells originated from one of the followings; mucous carcinoma, round cell carcinoma, locally advanced tumor, metastatic cancer, Ewing sarcoma, cancer metastasis, lymphatic metastasis, squaous cell carcinoma, esophageal squaous cell carcinoma, oral carcinoma, multiple myeloma, acute lymphoblastic leukemia, acute nonlymphoid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, effusion lymphoma, thymus lymphoma lung cancer, small cell lung cancer, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, adrenal cortical carcinoma, ACTH-producing tumor, non-small cell lung cancer, breast cancer, small cell carcinoma, ductal carcinoma, stomach cancer, colon cancer, colorectal cancer, polyp related to colorectal cancer formation, pancreatic cancer, liver cancer, bladder cancer, primary surface bladder tumor, invasive metastatic cell bladder carcinoma, muscle-invasive bladder cancer, prostate cancer, colon cancer, kidney cancer, hepatocarcinoma, esophageal cancer, ovarian carcinoma, uterine cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, primary peritoneum neoplasm, uterine cervical carcinoma, vaginal cancer, pudendum cancer, uterine cancer, follicle solid tumor, testis cancer, penis cancer, renal cell carcinoma, brain cancer, head/neck cancer, neuroblastoma, brainstem glioma, glioma, metastatic tumor cell invasion in central nervous system, osteoma, osteosarcoma, malignant melanoma, human skin keratinocyte tumor progression, squaous cell carcinoma, thyroid cancer, retinoblastoma, neuroblastoma, mesothelioma, Wilm's tumor, gallbladder cancer, trophoblastic tumor, hemangiopericytoma, and Kaposi's sarcoma.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them respectively in each vector, and cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors also cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
The pharmaceutical composition of the present invention preferably includes the composition of the invention, which is the active ingredient of the pharmaceutical composition, by 10˜95 weight % for the total weight of the pharmaceutical composition. The composition of the present invention can include, in addition to the active ingredient, one or more effective ingredients having the same or similar function to the active ingredient.
The composition of the present invention can include any generally used carrier, diluent, excipient, or a combination of at least two of those. The pharmaceutically acceptable carrier can be any carrier that is able to deliver the composition of the present invention in living body without limitation, which is exemplified by the compounds described in Merck Index, 13th ed., Merck & Co. Inc., such as saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and a mixture comprising one or more of those components. If necessary, a general additive such as antioxidant, buffer, and bacteriostatic agent can be additionally added.
The present composition can be formulated by using generally used excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants.
The composition of the present invention can be formulated for oral administration or for parenteral administration. The formulation for oral administration is exemplified by tablets, pills, powders, granules, capsules, and troches. The solid formulations for oral administration are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be added. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives.
Formulations for parenteral administration can include injections such as sterilized aqueous solutions, water-insoluble excipients, suspensions, and emulsions.
Water insoluble excipients and suspensions can contain propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc.
Therefore, the present invention also provides a composition for gene transfer for the treatment of cancer comprising the recombinant retrovirus above.
However, the recombinant retrovirus above can be produced from the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector comprising virus Env gene, promoter, and thymidine kinase gene. At this time, the first and the second recombinant expression vectors can be included in the recombinant retrovirus independently or together.
The cancer herein can include the cancers described hereinbefore.
In this invention, the term “composition for gene transfer” indicates the composition that can carry a gene into a target cell.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them respectively in each vector, and cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors also cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a method for preparing a replicating retrovirus vector system comprising the following steps: 1) constructing the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and 2) constructing the second recombinant expression vector comprising virus Env gene, promoter, and thymidine kinase gene.
The vector system above has the characteristics mentioned above. For example, the thymidine kinase gene can activate the prodrug ganciclovir and the cytosine deaminase gene can activate the prodrug 5-fluorocytosine.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them separately in each vector. The inventors cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors further cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a method for preparing a replicating retrovirus vector system comprising the following steps: 1) constructing the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and thymidine kinase gene; and 2) constructing the second recombinant expression vector comprising virus Env gene, promoter, and cytosine deaminase gene.
The vector system above has the characteristics mentioned above. For example, the thymidine kinase gene can activate the prodrug ganciclovir and the cytosine deaminase gene can activate the prodrug 5-fluorocytosine.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them separately in each vector. The inventors cloned P2 promoter and thymidine kinase gene in the vector containing Gag and Pol genes. The inventors further cloned P2 promoter and cytosine deaminase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
Therefore, the present invention also provides a method for treating cancer containing the step of administering the recombinant retrovirus above to a subject in need of the same.
The cancer can have the characteristics mentioned above. The subject can be mammals, and more specifically can be human. However, the recombinant retrovirus above can be produced from the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector comprising virus Env gene, promoter, and thymidine kinase gene. At this time, the first and the second recombinant expression vectors can be included in the recombinant retrovirus independently or together.
The recombinant retrovirus of the present invention can be administered via oral administration or parenteral administration according to the purpose of the administration and parenteral administration pathway can be selected from the group consisting of skin external application, intraperitoneal injection, intrarectal administration, hypodermic injection, intravenous injection, intramuscular injection, or intrathoracic injection.
The recombinant retrovirus of the present invention is preferably administered at an effective dose which can be determined by considering types of disease, severity of disease, drug activity, drug sensitivity, administration period, administration pathway, discharge rate, treatment period, and other drugs co-treated, etc. The composition of the present invention can be administered alone or together with other drugs. If co-treatment is needed, the administration could be performed stepwise or simultaneously.
However, to obtain a preferred effect, the concentration of the active ingredient in the composition of the present invention is 0.001˜10,000 mg/kg, and more preferably 0.1˜5 g/kg. The composition can be administered once a day or a few times a day.
In one embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them respectively in each vector, and cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors also cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
In addition, the present invention provides a use of the retrovirus above for the production of a drug for the prevention or treatment of cancer.
The cancer can have the characteristics mentioned above. The recombinant retrovirus above can be produced from the first recombinant expression vector comprising MuLV Gag-Pol gene, promoter, and cytosine deaminase gene; and the second recombinant expression vector comprising virus Env gene, promoter, and thymidine kinase gene. At this time, the first and the second recombinant expression vectors can be included in the recombinant retrovirus independently or together.
In another embodiment of the present invention, the inventors separated MuLV-Gag gene and MuLV-Pol gene, and GalV-EnV gene in order to express them respectively in each vector, and cloned P2 promoter and cytosine deaminase gene in the vector containing Gag and Pol genes. The inventors also cloned P2 promoter and thymidine kinase gene in the vector containing Env gene, leading to the construction of the replicating retrovirus vector (see
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
<1-1> Virus Vector Construction
In the conventional RRV vector, gag, pol, and MuLV-env genes are synthesized as one genome. Herein, the inventors induced the expression of gag-pol and MuLV-env separately in independent vectors. The env gene has been replaced with the GaLV (Gibbon ape Leukemia virus) env gene which has affinity to primates infection. The HSV-TK gene was cloned in gag-pol vector or GalV-env vector. A fluorescent gene was cross-cloned in each vector as a target marker, leading to the construction of the vector system.
Overall, the virus vector was constructed in the same method ing
1. spRRVe-P1-TK vector: spRRVe-P1-mcs vector having a multi-cloning site under promoter 1 (P1; MCMV immediate-early promoter) was digested with the restriction enzyme PmeI and then treated with CIAP (alkaline phosphatase, Calf intestinal). pSXLC-TK vector (Sugimoto et al., 1994, Bio/Technology 12, 694-698) was digested with the restriction enzymes NcoI and XhoI, and then TK gene was collected. The collected TK gene was treated with T4 DNA polymerase to make blunt end. spRRVe-P1-mcs vector was digested with the restriction enzymes BamHI and SalI, and then treated with CIAP. After collecting, cloning was performed. As a result, spRRVe-P1-TK vector was constructed (
2. sRRVgp-P1-RFP vector: sRRVgp vector (Korean Patent Publication No. 10-1381064) containing EcoRI cleavage site in between pol and 3′-LTR was digested with the restriction enzyme EcoRI, which was treated with CIAP. Lenti-P1-REP (monomer) vector (Chungnam National University, Korea) constructed by the present inventors was digested with the restriction enzymes PpuMI and NcoI and then P1-REP gene was collected. The collected P1-RFP gene was cloned in sRRVgp vector by using T4 DNA polymerase, leading to the construction of the vector (
3. sRRVgp-P1-TK vector: sRRVgp vector was digested with EcoRI and then treated with CIAP. A PCR product containing EcoRI, PmeI, and P1 promoter (EcoRI-P1-PmeI-EcoRI) was cloned in the vector sRRVgp, leading to the construction of sRRVgp-P1 vector. The vector was digested with the restriction enzyme PmeI, which was then treated with CIAP. TK gene was amplified so as to contain EcoRI cleavage site by using spRRVe-P1-TK vector (Chungnam National University, Korea) constructed by the present inventors as a template with the primer HSV-TK-EcoRI-F (5′-CGGAATTCATGGCTTCGTACCCCGGCCA-3′, SEQ. ID. NO: 1) and the primer HSV-TK-EcoRI-R (5′-GCGAATTCTCAGTAGCCTCCCCCATCTC-3′, SEQ. ID. NO: 2). The amplified gene was collected and digested with EcoRI, followed by cloning in sRRVgp-P1 vector (
4. spRRVe-P1-GFP vector: In order to clone GaLV-env gene in between gag gene and GFP gene of the retrovirus vector Retro-MCMV-GFP (MFG-mCMV-GFP, Korean Patent Publication No. 10-1381064) expressing GFP, Retro-P1-GFP vector (Chungnam National University, Korea) constructed by the present inventors was digested with the restriction enzyme PmeI. GaLV-env gene was amplified so as to contain PmlI cleavage site by using MYK-GaLV vector (Chungnam National University, Korea) constructed by the present inventors as a template with the primer GaLV-PmlI-F (5′-CGGCACGTGATGGTATTGCTGCCTGGG-3′, SEQ. ID. NO: 3) and the primer GaLV-PmlI-R (5′-GCCCACGTGTTAAAGGTTACCTTCGTT-3′, SEQ. ID. NO: 4). The amplified gene was collected and digested with PmlI, followed by cloning in Retro-P1-GFP vector (
<1-2> Virus Production
To produce virus with the vector constructed in Example <1-1>, spRRVe-P1-TK vector and sRRVgp-P1-RFP vector were combined together (spRRVe-P1-TK/sRRVgp-P1-RFP) and sRRVgp-P1-TK vector and spRRVe-P1-GFP vector were combined (sRRVgp-P1-TK/spRRVe-P1-GFP) together.
Overall, one day before the transfection, 293T cells were plated in a 6-well plate at the density of 6×105 cells/well. On the next day, the medium was replaced with FBS and antibiotics free 0.8 ml DMEM. The plate was placed in a cell culture incubator. In the meantime, DMEM was loaded in two 1.5 ml tubes (100 μl/tube) in order to prepare the solution for transfection. One tube contained 1 μg of RRV (spRRVe-P1-TK/sRRVgp-P1-RFP or sRRVgp-P1-TK/spRRVe-P1-GFP) DNA and 5 μl of PLUS reagent (Invitrogen), and the other tube contained 3 μl of lipofectamine (Invitrogen). The tubes stood at room temperature for 15 minutes after well mixing for 10 seconds. The solution containing lipofectamine was poured into the tube containing PLUS reagent, and mixed well for 10 seconds. The tube stood at room temperature for 20 minutes. The mixed solution comprising RRV DNA, PLUS reagent, and lipofectamine was loaded in the cell culture plate 208 μl/well slowly drop by drop, followed by culture for 4 hours. 4 hours later, the medium was discarded and DMEM supplemented with 3% FBS was loaded therein carefully not to make the cells fall off, followed by culture for 48 hours. 48 hours later, supernatant was obtained and filtered with 0.45 μm syringe filter. 30 ml of the filtrate was purified by using 15 ml centricon that can purify 100 kDa protein. To increase the purity, the obtained solution was filtered twice with 15 ml D-PBS, followed by 10-fold concentration. The solution was divided by 50 μl and stored in −80° C. deep freezer.
<1-3> Titration of the Produced Virus
Titer of the virus produced in Example <1-2> was measured by flow cytometry and qPCR.
Overall, one day before the virus infection, glioma cells (U87MG) were inoculated in a 6-well plate. On the next day, the virus 10-fold concentrated and separated was serially diluted in fresh medium (1×, 10×, 50×, 100×, and 500λ) but mostly by 10×. 1 ml of the diluted virus was added with 4 μg/10 of polybrene. The cell number was counted. Then, glioma cells were infected with the virus. 48 hours later, 50 uM of azidothymidine, the virus reverse transcription inhibitor, was added thereto, followed by reaction for 24 hours to inhibit the proliferation of the virus. GFP expression was quantified by flow cytometry and the titer was calculated by the following mathematical formula 1.
TU/ml=(cell number before infection×GFP ratio (%))/dilution ratio [Mathematical Formula 1]
However, the real-time PCR was performed by using a retrovirus titration set (Cat. #6166, Takara, Japan) according to the manufacturer's instruction to calculate the titer of the produced virus.
As a result, as shown in Table 1,
<1-4> Investigation of Recombination in the Replicating Retrovirus Vector
There is a high chance of recombination in the replicating retrovirus vector designed for gene therapy because the size of genomic RNA is increased due to the inserted foreign gene and the non-homologous nucleotide sequence added thereto. Therefore, in order to construct a stable and efficient replicating retrovirus vector for gene therapy, it is important to trace the chance of recombination in the course of construction and if any it is important to measure the level of recombination. To investigate the stability of the vector constructed in Example <1-1>, the following experiment was performed.
Overall, the glioma cells sub-cultured on the previous day were infected with 106 TU spRRVe-P1-TK/sRRVgp-P1-RFP or 106 TU sRRVgp-P1-TK/spRRVe-P1-GFP for three days. The supernatant was used to infect the U-87MG cells newly sub-cultured on the previous day 8 times stepwise at 3 days intervals. The resultant infected cells were named p1 (passage 1)˜p8 (passage 8). RFP or GFP expressed in each phase cells was observed under fluorescent microscope.
Therefore, PCR was performed with the genomic DNA extracted from the same phase cells using the Env vector and gag-pol vector specific primers, MuLV4194F (5′-AGCAAGCTATTGGCCACTG-3′, SEQ. ID. NO: 5), GaLV (1624)F(5′-GACTCAGTCAGCAAGTTAGAG-3′, SEQ. ID. NO: 6), and MFGSaclR (5′-CAATCGGAGGACTGGCGCCCCGAGTGA-3′, SEQ. ID. NO: 7), followed by confirmation of the gene amplification as expected. To the PCR reaction tube were loaded 100 ng of genomic DNA, 1× reaction buffer, 0.25 mM dNTP, 0.2 pmol of the forward primer, 0.2 pmol of the reverse primer, and 0.2 unit Taq polymerase and lastly sterilized distilled water was added to make the final volume 20 μl. PCR was performed according to the following conditions as listed in Table 1. The amplified DNA was loaded on 1% agarose gel, followed by analysis.
As a result, as shown in
<1-5> Analysis of the Recombination Pattern of spRRVe-P1-TK Vector
After PCR with the genomic DNA of the combined spRRVe-P1-TK/sRRVgp-P1-RFP in Example <1-4>, the PCR product obtained from p4 phase recombinant band (*) of spRRVe-P1-TK vector, which was smaller than expected, was collected and cloned in pGEM-T vector for gene analysis.
As a result, as shown in
<2-1> Virus Vector Construction
Since recombination did not occurred when TK gene was located in sRRVgp-P1 vector in Example <1-4>, another suicidal gene CD now being used in the clinical field as a treatment tool was inserted in spRRVe-P1 vector, leading to the construction of another vector system.
Overall, the virus vector was constructed as follows.
1. spRRVe-P1-yCD vector: pcDNA-yCD vector (Korea Cancer Center Hospital, provided by Dr. Lee) was digested with the restriction enzymes XhoI and HindIII. Then, yCD (SEQ. ID. NO: 16) was collected. The vector spRRVe-P1-MCS(Chungnam National University, Korea) constructed by the present inventors was digested with the restriction enzyme PmeI, where the yCD gene was cloned (
2. sRRVgp-P1-RFP vector: This vector was constructed by the same manner as described in Example <1-1> (
<2-2> Virus Production
To produce virus with the vector constructed in Example <2-1>, spRRVe-P1-yCD vector and sRRVgp-P1-RFP vector were combined together (spRRVe-P1-yCD/sRRVgp-P1-RFP), which was used to produce virus by the same manner as described in Example <1-2>.
<2-3> Investigation of Recombination in the Replicating Retrovirus Vector
The investigation was performed by the same manner and with the same conditions as described in Example <1-4> except that the infection with spRRVe-P1-yCD/sRRVgp-P1-RFP virus was performed 5 times stepwise at 3 days intervals to investigate the stability of the vector constructed in Example <2-1> and polymerization was induced at 72° C. for 90 seconds.
As a result, as shown in
<2-4> Analysis of the Recombination Pattern of spRRVe-P1-yCD Vector
After PCR with the genomic DNA of the combined spRRVe-P1-yCD/sRRVgp-P1-RFP in Example <2-3>, the PCR product obtained from p4 phase recombinant band (*) of spRRVe-P1-yCD vector, which was smaller than expected, was collected and cloned in pGEM-T vector. 12 clones were analyzed thereafter.
As a result, as shown in
<3-1> Virus Production
Virus was produced with the combined vector spRRVe-P1-yCD/sRRVgp-P1-TK by the same manner as described in Example <1-2> (
<3-2> Investigation of Recombination in the Replicating Retrovirus Vector
The investigation was performed by the same manner under the same conditions as described in Example <1-4> except that the infection with spRRVe-P1-yCD/sRRVgp-P1-TK was performed 5 times stepwise at 3 days intervals to investigate the stability of the vector used in Example <3-1>.
As a result, as shown in
<3-3> Analysis of the Recombination Pattern of spRRVe-P1-yCD Vector
After PCR with the genomic DNA of the combined spRRVe-P1-yCD/sRRVgp-P1-TK in Example <3-2>, the PCR product obtained from p4 phase recombinant band (*) of spRRVe-P1-yCD vector, which was smaller than expected, was collected and cloned in pGEM-T vector. Then, 10 clones proceeded to gene analysis.
As a result, as shown in
<3-4> Analysis of the Recombination Pattern of sRRVgp-P1-TK Vector
After PCR with the genomic DNA of the combined spRRVe-P1-yCD/sRRVgp-P1-TK in Example <3-2>, the PCR product obtained from p1 phase recombinant band (*) of sRRVgp-P1-TK vector, which was smaller than expected, was recovered and cloned in pGEM-T vector. Then, 20 clones proceeded to gene analysis.
As a result, as shown in
<3-5> Investigation of Drug Sensitivity of spRRVe-P1-yCD/sRRVgp-P1-TK Virus
The drug sensitivity against ganciclovir (GCV) and 5-fluorocytosine (5-FC) of the virus spRRVe-P1-yCD/sRRVgp-P1-TK produced in Example <3-1> was investigated.
Overall, 293T cells were co-transfected with spRRVe-P1-yCD/sRRVgp-P1-TK by using PLUS reagent (Invitrogen) and lipofectamine (Invitrogen). 2 days later, the supernatant was obtained. The U-87MG cells sub-cultured on the previous day in a 6-well plate at the density of 1.5×103 cells/well were added with 8 μl/ml of polybrene, followed by infection with the virus for 8 hours. 5 days after the infection (post-infection 5 d), the supernatant was obtained. The U-87MG cells sub-cultured on the previous day in a 6-well plate at the density of 1.5×103 cells/well were infected with the supernatant (p1). The infection was continued serially until p4 phase. The cells of each phase were treated with trypsin-EDTA to prepare single cells. The prepared single cells were distributed in a 12-well plate at the density of 1.5×103 cells/well. From the next day of the sub-culture, the cells were treated with 30 μl/ml of GCV and 1 mM 5-FC for 5 days or 8 days, followed by investigation of cell death.
As a result, as shown in
<4-1> Virus Vector Construction
Recombination in spRRVe-P1-yCD vector mainly occurred in between GaLV Env gene and P1 promoter and in between 3′-LTR and yCD gene, according to the investigation performed in Example <3-3>. So, the vector system was constructed by eliminating unnecessary nucleotide sequence for virus synthesis.
Overall, the virus vector was constructed with same method.
1. spRRVe-P1-CDa vector: Overlap PCR was performed to construct the vector using spRRVe-P1-yCD vector as a template with the primers spRRVe-CDa-F (5′-GAAGGTAACCTTTAATTCAATAACAGGAAAG-3′, SEQ. ID. NO: 8) and spRRVe-CDa-R (5′-CTTTCCTGTTATTGAATTAAAGGTTACCTTC-3′, SEQ. ID. NO: 9) (
2. spRRVe-P1-CDb vector: Overlap PCR was performed to construct the vector using spRRVe-P1-CD vector as a template with the primers spRRVe-CDb-F (5′-GAAGATATTGGTGAGTAGCTATAAAATAAAAGATTTT-3′, SEQ. ID. NO: 10) and spRRVe-CDb-R (5′-AAAATCTTTTATTTTATAGCTACTCACCAATATCTTC-3′, SEQ. ID. NO: 11) (
3. spRRVe-P1-CDc vector: Overlap PCR was performed to construct the vector using spRRVe-P1-CD vector as a template with the primers spRRVe-CDc-F (5′-ACCACCGTAGAACGCAATGGTGACAGGGGGAAT-3′, SEQ. ID. NO: 12) and spRRVe-CDc-R (5′-ATTCCCCCTGTCACCATTGCGTTCTACGGTGGT-3′, SEQ. ID. NO: 13)(
<4-2> Virus Production
Virus was produced with the combined vectors spRRVe-P1-CDa, spRRVe-P1-CDb or spRRVe-P1-CDc vector, and sRRVgp-P1-RFP vector; and spRRVe-P1-CDa, spRRVe-P1-CDb or spRRVe-P1-CDc vector, and sRRVgp-P1-TK vector constructed in Example <4-1> by the same manner as described in Example <1-2>.
<4-3> Investigation of Recombination in the Vectors spRRVe-P1-CDa, spRRVe-P1-CDb, and spRRVe-P1-CDc
The recombination in the vector was investigated by the same manner as described in Example <2-3> except that 6 kinds of viruses produced in Example <4-2> were used to infect the cells in order to investigate the stability of the vector constructed in Example <4-1>.
As a result, as shown in
<4-4> Analysis of the Recombination Pattern of spRRVe-P1-CDa Vector
After PCR with the genomic DNA of the combined vectors spRRVe-P1-CDa/sRRVgp-P1-RFP and spRRVe-P1-CDa/sRRVgp-P1-TK in Example <4-3>, the PCR product obtained from phase p2 and p3 recombinant bands (*) of spRRVe-P1-CDa vector, which was smaller than expected, was collected and cloned in pGEM-T vector. Then, 24 clones proceeded to gene analysis.
As a result, as shown in
The yCD gene used in Example 2˜Example 4 demonstrated recombination therein in the course of infection and also showed a low drug sensitivity against 5-FC. To overcome these disadvantages, 8 kinds of CD genes (CD2˜CD9) optimized by human codon were identified.
Overall, CD2 was developed from CD by codon optimization at Tocagen, which was used as the positive control. CD3 was the gene produced by optimizing 12 codons that were not optimized by human codon in CD2 gene. CD4 was the gene where 32 sites showing recombination of spRRVe-P1-yCD, spRRVe-P1-CDa, spRRVe-P1-CDb, and spRRVe-P1-CDc vector were all mutated. Therefore, 5 kinds of CD genes, CD5˜CD9, were the genes in which yeast CD was optimized by human codon. The 8 kinds of CD genes above were synthesized by Cosmogen Co., Ltd. The sequences of the CD2˜CD9 genes are as shown in table 3.
<6-1> Virus Vector Construction
A vector system was constructed with the CD gene optimized by human codon identified in Example 5.
Particularly, the virus vector was constructed as follows;
1. spRRVe-P1-CD2 vector: spRRVe-P1-RFF vector was digested with the restriction enzymes BamHI and ClaI and CD2 gene was cloned where RFP was eliminated (
2. spRRVe-P1-CD3 vector: spRRVe-P1-RFP vector was digested with the restriction enzymes BamHI and ClaI and CD3 gene was cloned where RFP was eliminated (
3. spRRVe-P1-CD4 vector: spRRVe-P1-RFP vector was digested with the restriction enzyme HpaI and CD4 gene was cloned where RFP was eliminated (
4. spRRVe-P1-CD5 vector: spRRVe-P1-mcs vector containing multi-cloning site under P1 region of spRRVe-P1 vector was constructed and digested with the restriction enzymes MluI and SalI, and CD5 gene was cloned therein (
5. spRRVe-P1-CD6 vector: spRRVe-P1-mcs vector containing multi-cloning site under P1 region of spRRVe-P1 vector was constructed and digested with the restriction enzymes MluI and SalI, and CD6 gene was cloned therein (
6. spRRVe-P1-CD7 vector: spRRVe-P1-mcs vector containing multi-cloning site under P1 region of spRRVe-P1 vector was constructed and digested with the restriction enzymes MluI and SalI, and CD7 gene was cloned therein (
7. spRRVe-P1-CD8 vector: spRRVe-P1-mcs vector containing multi-cloning site under P1 region of spRRVe-P1 vector was constructed and digested with the restriction enzymes MluI and SalI, and CD8 gene was cloned therein (
8. spRRVe-P1-CD9 vector: spRRVe-P1-mcs vector containing multi-cloning site under P1 region of spRRVe-P1 vector was constructed and digested with the restriction enzymes MluI and SalI, and CD9 gene was cloned therein (
<6-2> Virus Production
Virus was produced with the vectors spRRVe-P1-yCD, spRRVe-P1-CD2, spRRVe-P1-CD3, spRRVe-P1-CD4, spRRVe-P1-CD5, spRRVe-P1-CD6, spRRVe-P1-CD7, and spRRVe-P1-CD8 or the combined vector spRRVe-P1-CD9 and sRRVgp-P1-TK constructed in Example <6-1> by the same manner as described in Example <1-2>.
<6-3> Investigation of Drug Sensitivity of spRRVe-P1-CDs/sRRVgp-P1-TK Virus
The drug sensitivity of the virus produced in Example <6-2> against GCV and 5-FC was investigated by the same manner as described in Example <3-5> except that the virus produced in Example <6-2> was co-transfected.
As a result, as shown in
<6-4> Investigation of Recombination in spRRVe-P1-CDs
The stability of the vector used for the virus production in Example <6-2> was investigated by the same manner and under the same conditions as described in Example <1-4> except that the virus produced in Example <6-2> was infected 4 times stepwise at 3 days intervals and the polymerization was induced at 72° C. for 3 minutes and 20 seconds for which the primer GaLV (488)F (5′-GGCTAGAATCCCTATATGTA-3′, SEQ. ID. NO: 10) was used.
As a result, as shown in
<6-5> Analysis of the Recombination Pattern of spRRVe-P1-CDs
PCR products of p2 and p3 of spRRVe-P1-CD6 and spRRVe-P1-CD8 vector which had demonstrated excellent drug sensitivity against 5-FC in Example <6-3> were recovered (collected). The PCR products were cloned in pGEM-T vector. Then, 24 clones proceed to gene analysis.
As a result, as shown in
<7-1> Virus Vector Construction
It was confirmed from <Example 1>˜<Example 6> that the recombination in spRRV-P1 vector mainly occurred in P1 promoter region. This seemed because of the repeated nucleotide sequence of P1 promoter. So, P1 was replaced with P2 gene control region, leading to the construction of another vector system.
Overall, p1 promoter (MCMV immediate-early promoter) region was eliminated from each spRRVe-P1-CDs and p2 promoter (EF1α promoter) without repeated nucleotide sequence was cloned therein, leading to the construction of the vectors spRRVe-P2-yCD and spRRVe-P2-CDs. At this time, CD4 gene demonstrating a weak drug sensitivity was excluded (
<7-2> Virus Production
Virus was produced with the vectors spRRVe-P2-yCD, spRRVe-P2-CD2, spRRVe-P2-CD3, spRRVe-P2-CD5, spRRVe-P2-CD6, spRRVe-P2-CD7, and spRRVe-P2-CD8 or the combined vector spRRVe-P2-CD9 and sRRVgp-P1-TK constructed in Example <7-1> by the same manner as described in Example <1-2>.
<7-3> Investigation of Drug Sensitivity of spRRVe-P2-yCD/sRRVgp-P1-TK and spRRVe-P2-CDs/sRRVgp-P1-TK
The drug sensitivity of the virus produced in Example <7-2> against GCV and 5-FC was investigated by the same manner as described in Example <3-5> except that the virus produced in Example <7-2> was co-transfected to infect stepwise from p1 to p9 and cell death was observed after treating GCV and 5-FC for 9 or 12 days, respectively.
As a result, as shown in
<7-4> Investigation of Recombination in spRRVe-P2-yCD/sRRVgp-P1-TK and spRRVe-P2-CDs/sRRVgp-P1-TK
The stability of the vector constructed in Example <7-1> was investigated by the same manner and under the same conditions as described in Example <6-4> except that 8 kinds of viruses produced in Example <7-2> were used for the infection.
As a result, as shown in
<7-5> Analysis of the Recombination Pattern of sRRVgp-P1-TK
After PCR with the genomic DNA of the combined vectors spRRVe-P2-yCD/sRRVgp-P1-TK and spRRVe-P2-CDs/sRRVgp-P1-TK in Example <7-4>, the PCR product obtained from phase p3 and p4 recombinant bands of sRRVgp-P1-TK vector, which was smaller than expected, was recovered, followed by gene analysis.
As a result, as shown in
<8-1> Virus Vector Construction
As a result of Example 7, frequency of recombination in spRRVe-P2-CDs was lower than in the vector using P1 control region. However, gene loss was still observed in P1 control region in sRRVgp-P1-TK vector. The present inventors constructed sRRVgp-P2-TK vector system by replacing P1 promoter region with P2 promoter region.
Overall, PCR was performed to attach EcoRI, NotI, and PmeI restriction enzyme sequences to P2 promoter (EcoRI-P2-NotI-PmeI-EcoRI). The PCR product was digested with EcoRI, and inserted under Pol gene of sRRVgp vector (Chungnam National University, Korea) constructed by the present inventors, leading to the construction of sRRVgp-P2 vector. HSV-TK gene was amplified by PCR so as to contain NotI and PmeI restriction enzyme sequences (NotI-HSV TK-PmeI). Then, the PCR product was digested with NotI and PmeI, followed by cloning in under P2 gene. As a result, the vector sRRVgp-P2-TK was constructed (
<8-2> Virus Production
Virus was produced with the vector spRRVe-P2-CD2, the recombination free vectors spRRVe-P2-CD6 and spRRVe-P2-CD7 or the combined vector spRRVe-P2-CD8 and sRRVgp-P2-TK by the same manner as described in Example <1-2>.
<8-3> Investigation of Drug Sensitivity of spRRVe-P2-CDs/sRRVgp-P2-TK
The drug sensitivity of the virus produced in Example <8-2> against GCV and 5-FC was investigated by the same manner as described in Example <3-5> except that the virus produced in Example <8-2> was co-transfected to infect stepwise from p1 to p11 and cell death was observed after treating GCV and 5-FC for 5 or 7 days.
As a result, as shown in
<8-4> Investigation of Recombination in spRRVe-P2-CDs/sRRVgp-P2-TK
The stability of the vector constructed in Example <8-1> was investigated by the same manner and under the same conditions as described in Example <6-4> except that 4 kinds of viruses produced in Example <8-2> were infected 7 times stepwise at 3 days intervals.
As a result, as shown in
<8-5> Analysis of the Recombination Pattern of spRRVe-P2-CD6 and spRRVe-P2-CD8
PCR products of p7 of spRRVe-P2-CD6 and spRRVe-P2-CD8 confirmed to have recombination in Example <8-4> were collected. The PCR products were cloned in pGEM-T vector. Then, 19 clones proceed to gene analysis.
As a result, as shown in
In the examples above, CD6, CD7, and CD8 genes confirmed to have excellent drug sensitivity, compared to yCD gene, were identified. To secure CD gene that has maximized drug dependent cell killing activity, the present inventors additionally identified 7 other CD genes (CD10˜CD16) optimized by human codon.
7 kinds of CD genes, CD10˜CD16, were the genes in which yeast CD was optimized by human codon. The 7 kinds of CD genes above were synthesized by Cosmogen Co., Ltd. The sequences of the CD10˜CD16 genes are as shown in table 4.
<10-1> Virus Vector Construction
The CD genes optimized by human codon, newly identified in <Example 9>, were cloned in spRRVe-P2 vector, resulting in the construction of the vectors spRRVe-P2-CDs.
<10-2> Virus Production
Virus was produced by the same manner as described in Example <1-2> by using the combination of 7 kinds of spRRVe-P2-CDs constructed in Example <10-1> and sRRVgp-P2-TK vector.
<10-3> Investigation of Drug Sensitivity of spRRVe-P2-CDs/sRRVgp-P2-TK
The drug sensitivity of the virus produced in Example <10-2> against GCV and 5-FC was investigated by the same manner as described in Example <3-5> except that the virus produced in Example <10-2> was co-transfected to infect stepwise from p1 to p5 and cell death was observed after treating GCV and 5-FC for 6 or 8 days, respectively.
As a result, as shown in
<11-1> Virus Vector Construction
As serial infection phases were extended in Example <8-4>, even the vector containing P2 promoter, spRRVe-P2-CDs, displayed recombination action. To improve this problem, the combined vector, spRRVe-P2-TK/sRRVgp-P2-CDs, was prepared to construct a virus vector system.
Overall, the virus vector was constructed with same method.
1. spRRVe-P2-TK vector: PCR was performed using TK gene as a template with the primers HSV-TK-BamHI-F (5′-CGGGATCCATGGCTTCGTACCCCTGCCAT-3′, SEQ. ID. NO: 11) and HSV-TK-SalI-R (5′-CGGTCGACTCAGTTAGCCTCCCCCATCTC-3′, SEQ. ID. NO: 12). The PCR product was digested with the restriction enzymes BamHI and SalI, and then cloned in BamHI-SalI site of spRRVe-P2-mcs vector, leading to the construction of spRRVe-P2-TK vector (SEQ. ID. NO: 13) (
2. sRRVgp-P2-yCD vector: PCR was performed using yCD gene as a template with the primers CD-NotI-F (5′-CGGCGGCCGCATGGTGACAGGGGGAATGGC-3′, SEQ. ID. NO: 36) and CD-PmeI-R (5′-CGGTTTAAACCTACTCACCAATATCTTCAAA-3′, SEQ. ID. NO: 37). The PCR product was digested with the restriction enzymes NotI and PmeI, and then cloned in NotI-PmeI site of sRRVgp-P2 vector, leading to the construction of sRRVgp-P2-yCD vector.
3. sRRVgp-P2-CD2 vector: PCR was performed using CD2 gene as a template with the primers CD2-NotI-F (5′-CGGCGGCCGCATGGTGACCGGCGGCATGGC-3′, SEQ. ID. NO: 38) and CD2-PmeI-R (5′-CGGTTTAAACTTACTCGCCGATATCCTCGA-3′, SEQ. ID. NO: 39). The PCR product was digested with the restriction enzymes NotI and PmeI, and then cloned in NotI-PmeI site of sRRVgp-P2 vector, leading to the construction of sRRVgp-P2-CD2 vector.
4. sRRVgp-P2-CD6 vector: PCR was performed using CD6 gene as a template with the primers CD6-NotI-F (5′-CGGCGGCCGCATGGTTACTGGAGGGATGGC-3′, SEQ. ID. NO: 40) and CD6-PmeI-R (5′-CGGTTTAAACTCATTCGCCAATATCCTCGA-3′, SEQ. ID. NO: 41). The PCR product was digested with the restriction enzymes NotI and PmeI, and then cloned in NotI-PmeI site of sRRVgp-P2 vector, leading to the construction of sRRVgp-P2-CD6 vector (SEQ. ID. NO: 14) (
5. sRRVgp-P2-CD7 vector: PCR was performed using CD7 gene as a template with the primers CD7-NotI-F (5′-CGGCGGCCGCATGGTAACTGGTGGCATGGC-3′, SEQ. ID. NO: 42) and CD7-PmeI-R (5′-CGGTTTAAACCTACTCTCCGATATCCTCAA-3′, SEQ. ID. NO: 43). The PCR product was digested with the restriction enzymes NotI and PmeI, and then cloned in NotI-PmeI site of sRRVgp-P2 vector, leading to the construction of sRRVgp-P2-CD7 vector.
6. sRRVgp-P2-CD8 vector: PCR was performed using CD8 gene as a template with the primers CD8-NotI-F (5′-CGGCGGCCGCATGGTTACTGGGGGAATGG-3′, SEQ. ID. NO: 44) and CD8-PmeI-R (5′-CGGTTTAAACTTATTCCCCGATGTCTTCGA-3′, SEQ. ID. NO: 45). The PCR product was digested with the restriction enzymes NotI and PmeI, and then cloned in NotI-PmeI site of sRRVgp-P2 vector, leading to the construction of sRRVgp-P2-CD8 vector.
<11-2> Virus Production
Virus was produced by the same manner as described in Example <1-2> by using the combination of the vectors such as sRRVgp-P2-yCD, sRRVgp-P2-CD2, sRRVgp-P2-CD6, sRRVgp-P2-CD7, or sRRVgp-P2-CD8 constructed in Example <11-1> and spRRVe-P2-TK vector.
<11-3> Investigation of Recombination in spRRVe-P2-TK/sRRVgp-P2-CD (2,6,7,8)
The stability of the vectors constructed in Example <11-1> was investigated by the same manner and under the same conditions as described in Example <1-4> except that spRRVe-P2-TK/sRRVgp-P2-CD (2,6,7,8) were infected 7 times stepwise at 3 days intervals and polymerization was induced at 72° C. for 3 minutes.
As a result, as shown in
<11-4> Analysis of the Recombination Pattern of spRRVe-P2-TK/sRRVgp-P2-CDs
After PCR with the genomic DNA of the combined vectors spRRVe-P2-TK/sRRVgp-P2-yCD, spRRVe-P2-TK/sRRVgp-P2-CD2, spRRVe-P2-TK/sRRVgp-P2-CD6, spRRVe-P2-TK/sRRVgp-P2-CD7, and spRRVe-P2-TK/sRRVgp-P2-CD8 in Example <11-3>, the PCR products obtained from phase p6 and p7 of spRRVe-P2-TK, sRRVgp-P2-yCD, and sRRVgp-P2-CD (2,6,7,8) vectors were recovered, followed by gene analysis.
As a result, as shown in
<11-5> Investigation of Drug Sensitivity of spRRVe-P2-TK/sRRVgp-P2-CDs
The drug sensitivity of the virus produced in Example <11-2> against GCV and 5-FC was investigated by the same manner as described in Example <3-5> except that the virus produced in Example <11-2> was co-transfected to infect stepwise from p1 to p9 and cell death was observed after treating GCV and 5-FC for 8 or 12 days, respetively.
As a result, as shown in
This application is a continuation of PCT/KR2016/013881, filed Nov. 29, 2016, the contents of which is incorporated herein by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20180147299 A1 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2016/013881 | Nov 2016 | US |
| Child | 15365117 | US |
