Patent Application
20230250451
The present invention relates to the fields of tumor treatment and gene therapy in medical technology field, in particular to use of methioninase gene therapy in the targeting treatment of malignant tumors.
Malignant tumor is a common disease that seriously threatens human life and health. According to the latest statistics in 2015, the mortality rate of malignant tumor accounts for 23.91% of all deaths of residents. In the past 10 years, the incidence of malignant tumors in China has an annual growth rate of 3.9%, and the mortality rate has an annual growth rate of 2.5%. That is, more than 10000 people are diagnosed with cancer every day and 7.5 people are diagnosed with cancer every minute. Medical expenses caused by malignant tumors exceed 220 billion, and the situation of prevention and control is severe. At present, there are many means and methods to treat malignant tumors, such as surgery, chemical drug therapy, radiotherapy, etc. These treatment methods have a certain effect on malignant tumors, but always have serious adverse reactions, limited efficacy and are easy to develop drug resistance, etc. Therefore, the key is to seek new targeting and precision medicine such as gene therapy, for the treatment of malignant tumors. Due to the rapid growth of tumor cells, the demand for nutrients and metabolism are different from those of normal undifferentiated cells. In order to maintain continuous proliferation, tumor cells have to adjust their metabolism and nutrient acquisition methods. A large number of basic and clinical trials have shown that new drugs can be developed for effectively inhibiting tumor growth by targeting the metabolism of tumor dependent amino acids and derivatives thereof, such as methionine, glutamine, glutamate, arginine, tryptophan, etc. Therefore, targeting metabolic regulation treatment is a new direction of tumor treatment.
Methionine dependence is a common feature of most tumor cells, such as breast cancer, prostate cancer, lung cancer, colon cancer, kidney cancer, bladder cancer, melanoma, glioma, etc., whereas normal cells do not exhibit methionine dependence and more serious malignant tumors show increased methionine dependence. Several experiments in vivo and vitro successively confirmed that direct consumption of a methionine deficient diet could delay tumor cell proliferation. However, long-term deficiency or insufficiency of dietary methionine will cause malnutrition and metabolic disorders, and can even exacerbate carcinogenesis by chronically DNA hypomethylating. Therefore, reducing methionine in vivo through the specific decomposition of methionine by methioninase MEGL can more effectively inhibit tumor cell growth or eliminate tumor cells. However, mammals do not express methioninase on their own, and exogenous giving has certain side effects and always evokes an immune reaction in the body. Therefore, exploring endogenously expressed methioninase is a better choice. The purpose of cancer gene therapy is to introduce therapeutic genes into tumor cells. These therapeutic genes introduced into target cells can correct the mutated gene, inhibit active oncogenes, or produce other properties on the cell, etc. Suitable exogenous therapeutic genes comprise, but are not limited to, immunotherapeutic genes, antiangiogenic genes, chemoprotective genes, and “suicide” genes, and they can be introduced into cells by utilizing modified viral vectors or nonviral methods (comprising electroporation, gene gun and lipid or polymer coating). Requirements for an optimal viral vector comprise the efficient ability to find a specific target cell and express the viral genome in the target cell. All these properties of viral vectors have been developed in the past few decades and extensively studied in biomedicine such as retrovirus, adenoviruses, adeno-associated viruses, and oncolytic viral vectors, etc.
Methionine belongs to the essential amino acid and, catalyzed by methionine adenosyltransferase, generates S-adenosylmethionine (SAM). SAM, also known as active methionine, is the most important direct methyl donor in the body and participates in the catalytic reaction of methyltransfer of DNA, proteins and other different substances in the body. Histone methyltransferase EZH2 is the active component of histone methyltransferase in polycomb repressive complex 2 (PRC2). It is involved in chromatin condensation by adding the active methyl group of SAM to histone lysine 27 (H3K27) of histone 3, thereby inhibiting the transcription of related genes (such as anti-oncogene). Studies found that EZH2 and histone H3K27 methylation are closely related to cancers. EZH2 was first found to have high expression in lymphomas, metastatic prostate and breast cancers, and to be associated with breast cancer invasion. In addition, EZH2 is overexpressed in many human malignancies, such as lung cancer, lymphoma, leukemia, pancreatic cancer, cervical cancer, intestinal cancer, liver cancer, gastric cancer, melanoma, kidney cancer and bladder cancer, and its expression level is significantly elevated in metastasized tumors, which is closely associated with poor prognosis of cancer patients. Preclinical models of drugs for targeting EZH2 show their ability to inhibit the progression of brain cancer and prostate cancer. Thus, EZH2 could serve as a potential drug target for treating cancer metastasis by reducing EZH2 expression and activity, thereby reducing the methylation of histones and enhancing the expression of anti-oncogene.
In the present application, viral vectors were constructed by inserting exogenous MEGL gene to express methioninase. The vectors inhibit the growth and metastasis of malignant tumors by inhibiting the expression and activity of methyltransferase EZH2.
In one aspect, the present application provides a viral vector, wherein an exogenous MEGL gene is inserted therein.
Further, the exogenous MEGL gene is a methionine γ-lyase gene (GenBank accession No. 14313.1).
Further, the vector the vector uses EF1A promoter.
Further, the vector carries a mcherry fluorescent protein.
Further, the exogenous MEGL gene consists of a sequence of SEQ ID No.1.
The vector is constructed by following steps of: subcloning a MEGL gene into a plasmid to obtain a MEGL expression plasmid; transfecting the MEGL expression plasmid and a helper plasmid into 293T cells; and collecting a supernatant, concentrating and purifying to obtain the viral vector.
Further, the MEGL expression plasmid and the helper plasmid are co-transfected into the 293T cells, and the helper plasmid is viral packaging helper plasmid.
Further, the MEGL expression plasmid carries a mCherry red fluorescent protein.
The vector is constructed by following steps of:
(a) constructing an entry vector using BP reaction, comprising: mixing a Gateway expression vector having a target gene attB1-MEGL-attB2 sequence with a donor vector having attP1-ccdB-attP2 sequence; adding a BP Clonase enzyme mixture containing Int and IHF, keeping at 25° C. for 1 h, and treating with a protease K at 37° C. for 10 min to generate an entry vector having target gene MEGL and an expression vector having a suicide gene; transforming the entry vector into Escherichia coli Stbl3, and identifying positive clones and performing sequencing validation;
(b) constructing a destination vector having two recombination sites attR1 and attR2 downstream of an expression regulatory element thereof, each of the recombination sites being 125 bp in length; and having a ccdB suicide gene between the attR1 and attR2; and
(c) constructing a final expression vector via LR reaction, comprising: mixing the entry vector and the destination vector, adding a LR Clonase enzyme mixture containing recombinant factors including Int, IHF and Xis, keeping at 25° C. overnight, performing transformation by treating with a proteinase K at 37° C. for 10 minutes to generate a fusion plasmid, attL1 sequence and the attR1 sequence being recombined, the fusion plasmid being decomposed into two new plasmids, obtaining a final expression vector of destination vector having the target gene; transforming the final expression vector into Escherichia coli Stbl3, and identifying positive clone plasmid and performing sequencing validation.
Further, the viral vector is a lentivirus vector.
In another aspect, the present application provides use of the above viral vector in the manufacture of a medicament for treating malignant tumors.
Further, the medicament directly kills tumor cells.
Further, the malignant tumor is glioma.
In another aspect, the present application provides use of the above viral vector in the manufacture of histone methyltransferase EZH2 inhibitor.
In another aspect, the present application provides a method for treating malignant tumors, comprising administering the above viral vector or the viral vector constructed by the method above to subjects in need.
Further, the malignant tumor is glioma.
The viral vector is selected from the group consisting of lentivirus vector, retrovirus vector, adenovirus vector and other viral vectors known or under development in the art, and preferably is lentivirus vector.
The malignant tumors comprise but are not limited to glioma, breast cancer, prostate cancer, pancreatic cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, laryngeal cancer, leukemia, lymphatic cancer, melanoma, uterine cancer, ovarian cancer, skin cancer, bronchial cancer, bronchiolar cancer, urethral cancer, renal cancer, oral cancer, vaginal cancer, bile duct cancer, bladder cancer or nasopharyngeal cancer. The above medicaments or inhibitors can be administered via a variety of ways, comprising but not limited to: oral administration, local administration, injection (comprising but is not limited to intravenous, peritoneal, subcutaneous, muscular, intratumoral, spinal administration), etc.
Compared with the prior art, the present invention has the following advantages:
The present invention provides a virus system for expressing methioninase. The virus system can inhibit the expression and activity of methyltransferase EZH2, thereby inhibiting the growth and metastasis of malignant tumors.
Compared with retroviruses and recombinant adenoviruses, the virus expression system has many the advantages. For example, the virus expression system can harbor large exogenous gene segments, allow stable and long-term gene expression, have high transfection efficiency, and not cause cellular immune response. At the same time, mCherry fluorescent protein has less cytotoxicity and is conducive to the observation of cell infection.
The present application will be further illustrated with reference to following embodiments. However, these embodiments are only for illustrating, rather than limitations to the present invention detailed in the claims.
The viral vector is purchased from Cyagen Biology. The expression vector is mainly constructed using Gateway cloning technology. Gateway technology comprises two reactions, BP reaction and LR reaction. The BP reaction uses a recombination reaction between an attB DNA segment or expression clone and an attP donor vector to create an entry clone. The LR reaction is a recombination reaction between an attL entry clone and an attR destination vector. The details are described as follows:
1) Construction of the entry vector via BP reaction: Gateway expression vector having a target gene (attB1-MEGL-attB2 sequence) is mixed with a donor vector having attP1-ccdB (suicide gene)-attP2 sequence, and a BP Clonase enzyme mixture containing Int and IHF is added thereto. The resulted is kept at 25° C. for 1 h and then treated with a protease K at 37° C. for 10 min, so that recombination of attP and attB sequences occurs, generating an entry vector having target gene (MEGL) and an expression vector having suicide gene. The entry vector is transformed into Escherichia coli Stbl3, and positive clones are identified and sequencing validation is performed.
2) The destination vector, for matching with the Gateway system, has two recombination sites attR1 and attR2 downstream of an expression regulatory element thereof. Each of the recombination sites is 125 bp in length; and there is a suicide gene ccdB between the attR1 and attR2.
3) Construction of final expression vector via LR reaction: Two plasmids, i.e. the entry vector and the destination vector, are mixed, and a LR Clonase enzyme mixture containing recombinant factors including Int, IHF and Xis is added thereto. The resulted is kept at 25° C. overnight and then treated with a proteinase K at 37° C. for 10 minutes to perform transformation, so that the attR2 and attL2 sequences are recombined to generate a fusion plasmid. The attL1 sequence is recombined with the attR1 sequence, and the fusion plasmid is decomposed into two new plasmids, obtaining a final expression vector of destination vector having the target gene. The target product is transformed into Escherichia coli Stbl3, and positive clone plasmids are identified and sequencing validation is performed.
1) Virus packaging: The day before transfection, 293T cells are seeded in a culture dish. The number of seeded cells should be as such to allow cells growth to reach 90%-95% confluency on the day of transfection. On the day of transfection, culture medium is removed from 293T cells, and 10 mL (10 cm culture dish) of a culture medium for virus packaging is added thereto. Calcium phosphate-DNA precipitation is prepared as follows: A) Calcium-DNA mixture: CaCl2) is added into a 5 mL sterile EP tube, then an auxiliary plasmid and target gene plasmid are added thereto and mixed well. B) The centrifuge tube containing the calcium-DNA mixture is placed on a vortex oscillator to vortex the liquid, and then 2×HBS is added dropwise. After the dropping is completed, vortex for a few seconds and let stand for 5 minutes. The calcium phosphate-DNA suspension is poured into the culture medium of the above cells and mixed well gently, and placed in a 37° C., 5% CO2 saturated humidity incubator for culturing; After 4-6 hours of transfection, the culture medium is removed, and 10 mL of a culture medium for virus packaging is added. Then culture is continued in a 37° C., 5% CO2 saturated humidity incubator.
2) Collecting and concentrating virus: 48 h after transfection, the culture medium containing virus is collected into a 50 mL centrifuge tube; The virus supernatant was centrifuged at low speed to remove cell fragments. The resulted supernatant is collected and filtered with a 0.45 μM small filter, and the filtrate is collected. PEG6000 and NaCl solution are added thereto in an amount corresponding to the volume of filtrate and mixed well, placed at 4° C. overnight for precipitation, and centrifuged the next day at 4° C., 1500×g for 30 minutes. The supernatant is removed; The virus precipitate is dissolved with HBSS, and fully pipetted up and down to prepare a single virus suspension which is then sub packaged into cryogenic vials.
3) Identifying virus titer via Real time PCR: The day before the virus infection, 293T are seeded in a 6-well plate, five plates for each well, and 5×105 cells/well; After 24 hours of the seeding, cells from two wells are counted with a hemocytometer to determine the actual number of cells at the time of infection, which is recorded as N. The medium in other culture plates are discarded and replaced with fresh culture medium to a final concentration of 5 μg/mL polybrene. The concentrated virus is diluted 200 times with medium, that is, 1 μL virus is added to 199 μL medium. 0.5 μL, 5 μL and 5 μL diluted virus are added to three culture wells, respectively; 20 hours after infection, culture supernatant is removed and replaced with 500 μL fresh culture medium having DNaseI. Digest at 37° C. for 15 minutes to remove the residual plasmid DNA. The cells are then cultured in 2 mL normal medium for 48 hours, digested with 0.5 mL 0.25% trypsin-EDTA solution, and collected by centrifugation. Extract genomic DNA according to the instructions of DNeasy kit and conduct real time fluorescent PCR amplification. Titer (integration units per mL, IU/mL) is calculated according to formula as follows:
IU/mL=(C×N×D×1000)/V
wherein: C=average copy number of virus integrated per genome; N=number of cells at the time of infection (approximately 1×106) D=dilution ratio of viral vector; V=volume of diluted virus added.
1. Glioma cells U87 and snb19 in logarithmic growth phase are inoculated into a 6-well plate. When the cells are grown to a density of about 30%-50%, a control no-load viral vector (hereinafter defined as Group V) and MEGL overexpression lentivirus (hereinafter defined as Group M) are added according to MOI=10, and Polyprene was added as an aid to a final concentration of 5 μg/mL. After 24 h, replace with normal medium and continue culture for 48 h. Screen with Puromycin having a final concentration of 2 μg/mL. Fluorescence intensity and proportion of cells are observed under a fluorescence microscope every 24 hours. The screen is successful when the number of red fluorescence cells exceeds 95%. Cell screening results are shown in
2. Real time PCR and Western Blot for identifying the expression of methioninase in cells infected with MEGL virus.
Total RNA is extracted from well-grown cells after virus infection using Trizol method. 1 μg of the total RNA template is reverse transcribed into cDNA in vitro. With cDNA as template, amplification is completed after 30 cycles of pre-denaturation (94° C., 2 min), denaturation (94° C., 30 s), annealing (55° C., 30 s), elongation (72° C., 1 min), and a final elongation (72° C., 10 min). The amplification products are analyzed by agarose gel electrophoresis, and images are captured using the gel imaging system.
Wherein, the MEGL primer sequence is as follows:
The results are shown in
3. Identifying the Methioninase Activity of Cells Infected with MEGL Overexpression Virus. Intracellular Methionine Levels in Malignant Tumor Cells Infected by MEGL Overexpression Virus are Determined with Liquid Chromatography Mass Spectrometry (LC-MS).
Virus infected glioma cells U87-V, U87-M, snb19-V and snb19-M in the logarithmic growth phase are prepared into single-cell suspensions respectively at a concentration of 1.5×106/ml. 1 ml single-cell suspension is cultured in a 150 mm culture dish for 3 days and digested with trypsin. The treated cells are harvested and washed twice with pre-cooling PBS. 2 mL of pre-cooling 60% methanol aqueous solution is added to carry out extraction. Cells are broken up by sonication 5 times for 3 s, with 3 s interval between each sonication, operated on an ice bath. Centrifugation is carried out, the supernatant is collected into a sample vial, and the precipitate is extracted with 1 ml of 60% methanol. The supernatants are combined, freeze-dried, and left overnight. The resulted is re-dissolved in 300 ul pre-cooling 60% methanol aqueous solution, vortex sonicated, filtered with a 0.22 μm membrane, and detected. The results are shown in
Single-cell suspensions are prepared with virus infected glioma cells U87-V, U87-M, snb19-V and snb19-M in the logarithmic growth phase, and seeded in a 96-well plate, with 100 μL cell suspension (containing 1.5×103 cells) per well, 5 duplicates for each group; The proliferation of cells is detected on the 1st, 3rd, 4th, 5th and 7th days respectively as follows: 10 ul CCK-8 solution is added to each well without generating bubbles; The cells are incubated in an incubator at 37° C. with 5% CO2 for 1 h, then the culture plate is taken out, and the absorbance of the cells at 450 nm is measured with a microplate reader. Results shown in
Total RNA is extracted from well-grown cells after virus infection by using Trizol method. 1 μg RNA template is reverse transcribed into cDNA in vitro. Using cDNA as template, real time PCR is carried out at following reaction condition: 50° C. for 2 min; 95° C. for 10 min; 95° C. for 15 sec, 60° C. for 1 min; 72° C. for 1 min (40 cycles in total), and finally a product dissolution curve is measured, and calculation is carried out using 2 AAct method. Results shown in
The above in vitro experiments further show that the virus mediated methioninase system in present invention can inhibit the proliferation of tumors by inhibiting EZH2, and has good anti-tumor effects.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010591468.8 | Jun 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/105381 | 7/29/2020 | WO |
