Patent Application
20220364112
The present invention relates to a development of an oncolytic virus for treating brain tumors and a composition for treatment and a therapeutic agent using the same, and more particularly, to an oncolytic virus for treating brain tumors using a recombinant Newcastle disease virus into which a Newcastle disease virus vector (NDV)-based PTEN (phosphatase and tensin homolog) gene is inserted and a composition for treating brain tumors using the same.
Glioblastoma or brain tumor is the most common and serious symptom of cancer derived and generated from glial cells (glia, neuroglia) in the brain. It is also a serious cancer that occurs in the central nervous system. The general treatment used for such glioblastoma or brain tumor is a combination of surgery and methods such as chemotherapy or radiation therapy. The most common clinical symptoms that occur in patients are persistent headache, vomiting, aniseikonia, loss of taste, sudden personality change, dizziness, brain hemorrhage, etc. Various symptoms may occur depending on the area of the brain tissue affected by the cancerous tissue. It is known that more than 75% of malignant brain neoplasms are brain tumors or glioblastomas. The stage of malignant brain tumor and the shape of the brain tumor are derived from glial cells and are affected by how and in what form they are formed in the brain tissue. For these reasons, an effective therapeutic agent for each patient has not yet been developed.
Oncolytic virus treatment is taking a new approach as a new biopharmaceutical for cancer treatment and has made significant progress at experimental and clinical levels. Recently, the oncolytic virus used for virus treatment uses a method that uses the virus itself having oncolytic properties, a method that uses a virus that specifically acts on cancer cells and inserts a gene that has an effect on killing cancer cells, or both methods.
Accordingly, studies on various cancer treatment effects using Newcastle Disease Virus (NDV) are in progress. NDV is known to have an oncolytic effect that induces cancer cell death by actively proliferating only in cancer cells by itself. It is known that interferon-α induced in normal cells has an oncolytic effect due to the very weak or non-responsive properties in cancer cells. According to recent studies, it has been found to have a cancer cell killing effect by inducing an immune response to cancer cells by a specific viral protein and enhancing the apoptosis effect. When NDV infects normal cells, it has been found that the RNA genome of the NDV is easily destroyed by an immune response by interferon-a of the infected cells and thus does not proliferate, so it is not infective to mammals and does not form antibodies by the virus. These characteristics of NDV can be an advantage as a cancer therapeutic agent using a virus. In the 1950s, the cancer cell killing effect was tested using a velogenic NDV strain, and in certain cases, a clinical trial was also conducted for cancer patients. Various studies and clinical trials have provided a lot of evidence that cancer treatment using NDV has good prospects, and NDV does not infect normal mammalian cells, and maintains an effect on killing cancer cells or inhibiting growth of cancerous tissue without the side effects of repeated NDV inoculation. Also in clinical trials, there were cases where cancer patients survived for more than 5 years after being vaccinated with NDV for 5 years. However, a velogenic NDV is a deadly virus that infects chickens and is very dangerous for the poultry industry. Hence, there was a limit to the development of a cancer therapeutic agent with a velogenic virus.
In the 2000s, many scientists started developing an oncolytic virus using a mesogenic NDV. However, as the pathogenicity of the mesogenic NDV was reduced, it was identified that the efficacy was lower than that of the velogenic NDV in terms of cancer cell killing effect or cancer tissue growth inhibitory effect. The main reason for the low efficacy is that the mesogenic NDV has the same nature as a lysogenic virus and has a disadvantage that re-infection does not easily occur from an infected cancer cell to another cancer cell. Nevertheless, the biggest advantage of continuing to use NDV for treating brain tumors is that NDV can pass through the blood-brain barrier (BBB) through the nervous system infection, spread to and infect the brain, thereby infecting brain tumor cells, and killing brain tumor cells. In order to overcome these shortcomings of mesogenic NDV, a recombinant NDV containing various cancer cell death and cancer tissue growth inhibitory genes has been developed. The recombinant NDV into which the tumor inhibitory gene is inserted shows better efficacy than the existing mesogenic NDV and continues to develop through various studies related to NDV.
PTEN (phosphatase and tensin homolog) is a well-known tumor inhibitory gene and is an enzyme that removes 3′ phosphate of PIP3 (Phospho-inositol triphosphate) and converts the same into PIP2 (phospho-inositol biphosphate). It regulates cell migration and viable cell proliferation through dephosphorylation. In addition, it is known that the enzyme activity of PTEN is lost due to deletion or mutation of the PTEN gene, thereby inhibiting apoptosis and actively promoting cell proliferation during cancer progression. Abnormalities in the PTEN gene are known to cause cancer, including prostate cancer, endoderm-derived cancer, breast cancer, lung cancer, etc. Recently, abnormalities in the phosphatidylinositol 3-OH kinase pathway due to PTEN abnormalities in more than 50% of brain tumor patients have been found to be an important cause. The attenuated phosphatase activity of PTEN is closely related to cancer suppression and is an important gene for cell cycle regulation and proliferation regulation. Accordingly, the present invention is to provide a method for developing a viral therapeutic agent for brain tumors by inserting the PTEN gene, which is known to cause brain tumors, into a recombinant NDV, and more specifically, to an oncolytic virus for treating brain tumors using a recombinant Newcastle disease virus into which a Newcastle disease virus vector (NDV)-based PTEN gene is inserted and a composition for treating brain tumors using the same.
An aspect of the present invention is to provide an LVP-K1 vector for inserting a foreign gene containing Newcastle Disease Virus (NDV) cDNA and a transgene cassette.
In addition, another aspect of the present invention is to provide a recombinant Newcastle disease virus containing the LVP-K1 vector for inserting the foreign gene and a tumor inhibitory gene PTEN (phosphatase and tensin homolog) gene.
In addition, another aspect of the present invention is to provide a pharmaceutical composition for prevention or treatment of brain tumors containing the recombinant Newcastle disease virus or a virus purified therefrom.
In addition, another aspect of the present invention is to provide a method for preventing or treating brain tumors including administering the recombinant Newcastle disease virus or a virus purified therefrom.
In addition, another aspect of the present invention is to provide a method for providing information on brain tumor prevention or treatment including administering a recombinant Newcastle disease virus or a virus purified therefrom.
In addition, another aspect of the present invention is to provide a method for producing a recombinant Newcastle disease virus containing the PTEN gene and capable of being expressed as a protein in cancer cells.
Finally, another aspect of the present invention is to provide a method for evaluating an effect of treating brain tumors in animals.
One aspect of the present invention provides an LVP-K1 vector for inserting a foreign gene containing a Newcastle Disease Virus (NDV) cDNA and a transgene cassette containing genes encoding NP, P, M, F, HN and L proteins as active ingredients, in which the transgene cassette consists of an IGS sequence (GE (gene end), IG (intergenic sequence) and GS (gene start)) and an MCS (multiple cloning site).
In one embodiment of the present invention, the “vector” may be represented by a nucleotide sequence represented by SEQ ID NO: 1 but is not limited thereto.
In one embodiment of the present invention, the “transgene cassette” may be inserted between an NP gene and a P gene of a Newcastle disease virus but is not limited thereto.
In one embodiment of the present invention, the “LVP-K1 vector” may further include a kozak sequence represented by a nucleotide sequence represented by SEQ ID NO: 6 but is not limited thereto.
In addition, the present invention provides an LVP-K1-PTEN vector further including a PTEN gene represented by a nucleotide sequence represented by SEQ ID NO: 3 in the LVP-K1 vector.
In one embodiment of the present invention, the “vector” may be represented by a nucleotide sequence represented by SEQ ID NO: 2 but is not limited thereto.
In one embodiment of the present invention, the “PTEN gene” may be introduced between the NP and P genes but is not limited thereto.
In addition, the present invention provides a recombinant Newcastle disease virus (Accession No.: KCTC 14422BP) into which the LVP-K1-PTEN vector is introduced (Depository Institution: Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Biotechnology and Bioscience; Accession No.: KCTC 14496BP; Deposit Date: 20210312).
In addition, the present invention provides a pharmaceutical composition for preventing or treating brain tumors containing a recombinant Newcastle disease virus (Accession No.: KCTC 14422BP) as an active ingredient.
In one embodiment of the present invention, the composition may further include an antigen purified from a recombinant Newcastle disease virus but is not limited thereto.
In addition, the present invention provides a method for preventing or treating brain tumors, in which the method includes administering the composition to an animal.
In addition, the present invention provides a method for providing information on brain tumor prevention or treatment, in which the method includes administering the composition to an animal.
In addition, the present invention provides a method for evaluating an effect of preventing or treating brain tumors in an animal, in which the method includes administering the composition to an animal.
In one embodiment of the present invention, the “method” may be measuring and evaluating changes in cancer cells or cancer tissues of animals but is not limited thereto.
Finally, the present invention provides a method for producing a recombinant Newcastle disease virus, in which the method includes: inoculating the recombinant Newcastle disease virus into a host cell line; culturing the host cell line; and obtaining the recombinant Newcastle disease virus from a culture of the host cell line.
It has been identified that it is possible to treat brain tumors through brain tumor apoptosis by expressing PTEN protein in cancer cells when a PTEN gene is included in a Newcastle disease virus (NDV) that can spread to the human brain for the purpose of treating brain tumors caused by abnormalities in the PTEN (phosphatase and tensin homolog) gene, which is identified to be more than 50% of the causes of brain tumors of the present invention. Accordingly, for the brain tumor treatment effect, the present invention developed LVP-K1-PTEN oncolytic virus to complete the development of a safe and effective brain tumor treatment, which may contribute to treatment of patients with brain tumors and brain tumor treatment by inhibiting the deterioration of brain tumors.
Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following examples are provided by way of illustration of the present invention. When it is determined that the specific description of known techniques or configuration well known to those skilled in the art unnecessarily obscure the gist of the present invention, the detailed description therefor may be omitted, and the present invention is not limited thereto. The present invention allows various modifications and applications within the description of the claims to be described later and the scope of equivalents interpreted therefrom.
Further, terminologies used herein are terms used to properly represent preferred embodiments of the present invention. It may vary depending on the intent of users or operators, or custom in the art to which the present invention belongs. Accordingly, the definitions of these terms should be based on the contents throughout this specification. In the entire specification, when a part is referred to as “comprising” a component, it means that it may further include other components without excluding other components unless specifically described otherwise.
Throughout the specification, “%” used to refer to the concentration of specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid and (volume/volume) % for liquid/liquid, unless specified otherwise.
Hereinafter, the present invention will be described in more detail.
The present invention provides an LVP-K1 vector for inserting a foreign gene containing a Newcastle Disease Virus (NDV) cDNA and a transgene cassette containing genes encoding NP, P, M, F, HN and L proteins as active ingredients.
In addition, the transgene cassette consists of an IGS sequence (gene end (GE), intergenic sequence (IG), and gene start (GS)) and a multiple cloning site (MCS).
As used herein, the term “Newcastle disease virus (NDV)” belongs to a paramyxovirus having a (−) sense RNA genome of about 15 kb and is known as a safe virus for mammals without human infectivity. NDV genomic RNA has an extragenic leader sequence of about 30 bases and a tail sequence of about 50 bases. Two sequences at both termini are known to control the transcription and replication of viral genes and the encapsidation of newly synthesized RNA genomes into viral particles. The NDV gene configuration consists of six genes including NP, P, M, F, HN and L between both terminal leader and tail genes, and each gene encodes a nucleoprotein (NP), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutinin-neuraminidase protein (HN), and a large protein (L).
In the Newcastle disease virus, an IGS (GE-IG-GS) sequence exists between each gene, and each gene undergoes a transcriptional process in the initial stage of host cell infection to synthesize a movement protein to the endoplasmic reticulum (ER) of the host cell. Then, when the amount of M protein synthesis rises above a certain level, a (+) sense RNA genome is synthesized, and a (−) sense RNA genome is synthesized using this as a template. The finished virus particles are expelled out of the cell.
It is known that the “ability of the Newcastle disease virus to introduce foreign genes” is up to 6 kb, and the introduction of foreign genes has been mainly made between the P and M genes and between the HN and L genes. However, although it is known that all of the foreign genes may be introduced between the six genes, it is known that each location has an effect on mRNA expression, protein expression, and, in severe cases, virus proliferation. However, quantitative comparison tests for each location have not been performed. There is a GE-IG-GS gene between each gene. In particular, in the case of an IG gene, it is made up of 1 or 2 nucleotides between NP-P, P-M and M-F, 35 nucleotides between F-HN, and 47 nucleotides between HN-L. After virus infection, the (−) sense RNA genome synthesizes the mRNA of each protein at the initial stage of infection by the NP, P, and L proteins possessed by NDV, and the synthesized mRNA moves to the endoplasmic reticulum of the host cell to synthesize the protein of each gene. Thereafter, the (+) sense RNA genome is synthesized by the interaction of the NP, P, and L proteins with the M protein, and many copies of the (−) sense RNA genome are synthesized using this as a template and released out of the host cell. It is known that the amount of mRNA synthesis for autologous protein production at the time of initial infection is the most at the N-terminus, in other words, NP mRNA is synthesized the most, and the mRNA synthesis decreases as it moves away from the N-terminus afterward.
For cDNA construction of a Newcastle disease virus, a method of making the NDV (−) sense RNA genome into multiple fragments of double-stranded DNA through the reverse transcription polymerase chain reaction (RT-PCR) method, and then re-ligating each fragment to create a cDNA clone of the entire NDV is being used. In cDNA preparation using this method, point mutation is highly likely to occur due to the nature of reverse transcriptase, so after the cDNA is finished preparing, the gene sequence of 15 kb is identified through sequencing. When one or more point mutation occurs, a process of making cDNA from the NDV genome needs to be repeated again. Recombinant NDV is constructed by inserting the cDNA fragment into the pBR322 vector.
In addition, a transgene cassette was created and inserted so that a foreign gene may be easily introduced into a position into which the foreign gene may be inserted so that the antigen protein may be expressed or operated through a recombinant Newcastle disease virus. The transgene cassette is composed of a GE-IG-GS sequence and a multiple cloning site (MCS) in front of the N-terminus of the foreign gene insertion site and may be constructed by inserting the transgene cassette between the NP and P genes, between the P and M genes, and between the HN and L genes in compliance with the rule of six together with various restriction enzyme sequences. Preferably, the transgene cassette may be inserted between the NP and P genes, and between the P and M genes, and more preferably, the transgene cassette may be inserted between the NP and P genes.
According to one embodiment of the present invention, the LVP-K1 vector for inserting the foreign gene may construct a recombinant NDV virus through an overlap cloning method after putting the transgene cassette in a perfectly made recombinant NDV between each gene, dividing it into 4 fragments of DNA, ligating each NDV fragmented gene to pBR322 plasmid DNA, and then performing transformation into TOP10 E. coli to construct and store 4 types of recombinant strains, and then separating the gene from each recombinant E. coli strain when introducing a new gene, and obtaining a fragment of the gene using PCR, but is not limited thereto.
In addition, the vector constructed by the above method prevents point mutation that occurs during the process of making a recombinant Newcastle disease virus each time and has a feature that a foreign gene may be easily inserted into the NDV cDNA through a multiple cloning site (MCS).
In addition, the LVP-K1 vector for inserting the foreign gene may be composed of the nucleotide sequence represented by SEQ ID NO: 1 and includes a functionally equivalent substance thereto. The term “functionally equivalent substance” refers to a gene or gene combination including a sequence having at least 70%, preferably at least 80%, and more preferably at least 90% of homology with a gene sequence represented by SEQ ID NO: 1 as a result of a substitution or deletion of a nucleotide and exhibiting substantially identical physiological activity to the gene having the gene sequence represented by SEQ ID NO: 1.
Next, the present invention provides a recombinant Newcastle disease virus including the LVP-K1 vector for inserting the foreign gene and a gene encoding a human PTEN (phosphatase and tensin homolog) protein.
In addition, the “PTEN gene” is known to produce energy by converting glucose into lactic acid while rapidly growing cancer cells in brain cells. In this series of metabolic processes, the PTEN-PI3K-AKT-mTOR signaling system plays a central role in glucose metabolism and is related to increased ATP consumption due to glycosylation in glucose uptake and transport lipogenesis-inducing protein synthesis. A series of metabolic processes caused by abnormalities in the PTEN gene are being studied to be closely related to cancer cell growth. It is a gene established based on the PTEN gene information (Gene ID: NCBI Reference Sequence: NG_007466.2).
In addition, the “PTEN protein” refers to a protein expressed in cells of various human organs, and preferably may be a PTEN protein expressed in glial cells, and the exact amino acid sequence may be the sequence listed by SEQ ID NO: 4.
As for a method for securing the PTEN protein-coding gene, a polymerase chain reaction (PCR) method using either artificially synthesized using a gene synthesizer or a primer capable of complementary binding from the PTEN gene present in human cells may be used. Depending on the expression system, there may be differences from the gene encoding the human PTEN protein due to codon optimization. Therefore, the gene encoding the recombinant PTEN may exist in the form of various nucleotide sequences including amino acid residues of the PTEN protein.
The “host cell into which the PTEN protein gene is introduced” may be a prokaryotic or eukaryotic cell, and any cell having a high expression rate of the introduced PTEN protein may be used without limitation. Examples include E. coli, mammalian cell lines, insect cell lines, fungi, yeast, eukaryotic and prokaryotic host cells such as recombinant viruses, and the like.
In addition, “expression of PTEN protein” may be expressed as a simple subunit protein or may exist in a form that is exposed to the outside by binding to a specific virus or virus surface. Preferably, the gene is transferred to a host cell by a virus and expressed in a cell line. Preferably, the normal intracellular expression and functional activity of the PTEN protein transferred by the virus will be expressed to be maintained.
Examples of viruses that may be used for the expression of the PTEN protein include a lentivirus, a retrovirus, a vaccinia virus, an adenovirus, and an adeno-associated virus, a cytomegalovirus, a Sendai virus, a poxvirus, a Newcastle disease virus, and an alphavirus. Any virus capable of expressing a protein through introduction of a foreign gene and capable of producing high stability, high expression ability and high viral titer may be used without limitation. Preferably, it may include a poxvirus, a flavivirus, an alphavirus, and a Newcastle disease virus as an enveloped virus. More preferably, it may be a Newcastle disease virus (NDV) that is a safe virus without human infectivity and capable of producing a high viral titer.
The “Newcastle disease virus” is a legal infectious disease that infects chickens and causes neurological and respiratory symptoms and is a very lethal virus for chickens. According to the pathogenicity, it is divided into velogenic, mesogenic, and lentogenic Newcastle disease viruses, all of which may be used in the production of PTEN gene transfer virus vector vaccines, but preferably mesogenic and lentogenic viruses may be used. More preferably, it may be a recombinant virus using a lentogenic virus strain.
The transgene cassette expressing the PTEN gene, MCS, and NP and P genes of the NDV strains are genes or a combination of genes designed to exert the effect of inhibiting proliferation and killing of cancer cells when the PTEN protein gene is delivered to brain tumor cells through recombinant NDV and expressed as a normal protein. Accordingly, the overall combination from the NP protein to the transgene cassette, the PTEN gene, and the P gene functionally provides an optimal nucleotide sequence for transduction expression and functional activity of PTEN protein gene. Various nucleotide sequences having the same functional activity will be possible, and preferably 70% or more of the same nucleotide sequence. More preferably, it will be 80% or more of the same nucleotide sequence, and most preferably, it will be a nucleotide sequence having 90% or more of functional activity.
In addition, the “recombinant Newcastle disease virus (Accession No. KCTC14496BP)” may be one into which the LVP-K1-PTEN vector represented by the nucleotide sequence represented by SEQ ID NO: 2 is introduced and includes a functionally equivalent substance. The term “functionally equivalent substance” refers to a gene or gene combination including a sequence having at least 70%, preferably at least 80%, and more preferably at least 90% of homology with a gene sequence represented by SEQ ID NO: 2 as a result of a substitution or deletion of a nucleotide and exhibiting substantially identical physiological activity to the gene having the gene sequence represented by SEQ ID NO: 2.
In addition, the present invention provides a pharmaceutical composition for prevention or treatment of brain tumors containing the recombinant Newcastle disease virus or a purified virus as an active ingredient.
Since the pharmaceutical composition of the present invention includes the above-described recombinant Newcastle disease virus, the description of the contents overlapping with the above-described recombinant Newcastle disease virus of the present invention is omitted in order to avoid the excessive complexity of the present specification due to the overlapping description.
In addition, the pharmaceutical composition may further include an immune enhancing material or adjuvant. The immune enhancing material or adjuvant may be a material that can help the treatment effect by inducing an immune response of cancer cells but is not limited thereto.
The “recombinant Newcastle disease virus (Accession No. KCTC14496BP)” may be in any form known in the pertinent field, for example, a solid form suitable for solution and injectables or suspension but is not limited thereto. Such formulations may also be formulated into an emulsified or encapsulated form for easy absorption into the body, or in the form of an aerosol or spray. They may also be incorporated into transdermal patches. Liquids or injectables may contain propylene glycol if necessary and sodium chloride in an amount sufficient to prevent hemolysis (for example: about 1%).
In addition to the therapeutic virus of the present invention, a pharmaceutically acceptable carrier or diluent may be included. Herein, the term “pharmaceutically acceptable” refers to a non-toxic composition that is physiologically acceptable and does not inhibit an action of the active ingredient when administered to humans and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions.
The technical fields requiring viral stability and safety as therapeutic agents are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions, or emulsions. As the adjuvant, a typical or atypical organic or inorganic polymer or the like may be used. As the composition that may be added, stabilizers, antibiotics, preservatives, and the like may be used. Depending on the route of administration, the virus may be used by mixing with distilled water or a buffer solution.
The therapeutic virus may be administered via direct injection into cancer tissue or an administration route such as oral, intramuscular, subcutaneous, intravenous, etc., but is not limited thereto, and may preferably be administered through an intravenous route.
The term “subject” of the present invention refers to a subject in need of a method of control or treatment for the treatment of disease and alleviation of symptoms, and more specifically, a human, or a mammal such as a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow.
The term “treatment” of the present invention refers to all actions such as inhibition of proliferation or death of brain tumor cells by administration of a composition according to the present invention, and delay of increase or decrease in brain tumor tissue.
The term “treatment” of the present invention refers to all actions that alleviate or beneficially change symptoms for brain tumors by administration of a composition according to the present invention.
Moreover, the present invention provides a method for producing a recombinant Newcastle disease virus, in which the method includes: inoculating the recombinant Newcastle disease virus into a host cell line; culturing the host cell line; and obtaining the recombinant Newcastle disease virus from a culture of the host cell line.
In the present invention, the recombinant viruses LVP-K1-PTEN and LVP-K2-PTEN may be recovered through a conventional virus production method. After the production of infectious clone cDNA represented by SEQ ID NO: 2 for PTEN protein expression was completed, three types of helper plasmids (NP, P, L) and modified vaccinia virus (MVA/T7) were injected into the HEp-2 cell line and cultured, followed by the recovery of the recombinant virus according to a conventional method. Transfection was performed using lipofectamine 3,000 as an injection method into the cell line, and HEp-2 cells were used as the cell line. After culturing for 3 to 4 days, the recombinant virus was recovered and inoculated into the allantoic cavity of an 8 to 10 day old SPF embryonated egg. After culturing the virus, the allantoic fluid was recovered and the virus titer was increased by culturing at least two blind passages on embryonated eggs in the same way. After purification from allantoic fluid by a conventional purification method, it was cultured in Vero76 cells selected as an appropriate cell line and used in the experiment. As for a method for identifying the expression of the PTEN protein of the recombinant virus strains LVP-K1-PTEN and LVP-K2-PTEN virus, a reverse transcription PCR method was used to identify the gene stability of the PTEN protein (
Purification of the virus proceeds clarification by centrifugation after harvesting the recombinant virus culture medium. Clarification may be performed by centrifugation or microfiltration. The centrifugation may be performed under the conditions of 10,000 g, 10 minutes, and 4° C. so that supernatants may be used for the next purification process. In the case of microfiltration, a filter with a pore size of 1.0 μm to 0.2 μm may be used, and a filter with a pore size of 0.45 μm may be preferably used. As the filtration method, either dead end filtration or cross flow filtration may be used, and both methods are applicable. Recombinant virus purification is possible through known methods, including extraction through chromatography or ultrafiltration method. In the purification method using chromatography, virus purification is possible through a combination of an appropriate resin and buffer through a difference in binding power such as affinity, ion exchange, size exclusion, and hydrophobicity. Usually, virus purification is recovered by precipitating or separating the virus by ultra-high-speed centrifugation using sucrose gradient media, and the recovered virus is resuspended in THE buffer for use in the next process. Recombinant virus was purified using cation exchange resin chromatography, and after sample loading, the fraction extracted at a specific concentration was recovered through a sodium chloride concentration gradient. The recovered fraction was recovered by precipitating or separating the virus by ultra-high-speed centrifugation using sucrose gradient media, and the recovered virus was resuspended in physiological saline for injection and used in the next process.
In addition, the present invention provides a brain tumor treatment induction method including administering to a human a therapeutically effective amount of the viral composition in order to induce a treatment effect on a human brain tumor.
In addition, the treatment effect is demonstrated by a reduction or absence of clinical symptoms normally exhibited by a brain tumor, a faster recovery time or a lower duration, a difference in a low number of brain tumor cells in a sample of blood, body fluid or organ of brain tumor cells, a reduction of brain tumor tissue, and death of brain tumor cells.
In addition, the effective amount of a therapeutic agent refers to an amount capable of inducing a treatment effect and inducing effects such as a reduction of clinical symptoms caused by brain tumors in humans, reduction of cancer cells, reduction of cancer tissue, etc., and may be appropriately selected by those skilled in the art. For example, in the case of an effect amount of a therapeutic agent containing a recombinant viral composition, an amount of the purified virus may be 105.0 TCID50/ml to 1011.0 TCID50/ml. More preferably, it may be 108.0 TCID50/ml to 109.0 TCID50/ml or more.
The method for inducing a treatment effect is not limited thereto, but may be inoculating the composition through oral, transdermal, intramuscular, intraperitoneal, intravenous, or subcutaneous routes. Preferably, the first and second or more compositions may be directly injected into a vein or cancer tissue. More preferably, it may be an intravenous injection. There is no limit to the number of inoculations depending on a treatment effect.
In addition, the present invention provides a method for providing information on brain tumor prevention or treatment, in which the method includes administering a recombinant Newcastle disease virus to a subject.
In addition, the present invention provides a method for evaluating a treatment effect in an animal, in which the method includes administering a recombinant Newcastle disease virus to an animal.
In addition, the method may be to measure the cancer cell killing effect using a human cancer cell line.
The cancer cell line may be a brain tumor-derived cell line, preferably a T98G cell line. In addition, patient-derived cancer cell lines may be used, and the present invention is not limited only to brain tumor-derived cell lines.
The present invention provides a method of injecting the LVP-K1-PTEN virus into a cancer tissue of a mouse transplanted with brain tumor cells, that is, a xenograft model. After generation of cancer tissue, intravenous injection or direct injection into cancer tissue may be used, and a method for measuring the effect of virus injection, such as complete remission, partial remission, or reduction of cancer tissue, may be provided.
The “LVP-K1-PTEN virus” of the present invention was deposited with the Korean Collection for Type Cultures (KCTC) on Mar. 12, 2021, and was given an Accession No.: KCTC 14496BP.
Hereinafter, the examples of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only intended to embody the contents of the present invention, and the present invention will not be limited thereto.
NDV VG/GA has about 15 kb of negative-sense single-stranded RNA as genetic information and is composed of 6 ORFs, and the proteins that form a structure of the virus encode NP (nucleoprotein, SEQ ID NO: 7), P (phosphoprotein, SEQ ID NO: 8), M (matrix protein, SEQ ID NO: 9), F (fusion protein, SEQ ID NO: 10), HN (hemagglutinin-neuraminidase, SEQ ID NO: 11) and L (RNA-directed RNA polymerase, SEQ ID NO: 12) genes. After RNA isolation using a viral RNA extraction kit (Qiagen), four pairs of primers specific for genes were prepared and reverse transcription polymerase chain reaction (RT-PCR) was performed. Five pairs of primers specific to the gene are shown in Table 1 (showing the primers used during the insertion process using a restriction enzyme into a pBR322 vector after cDNA synthesis of the present invention). RT-PCR was performed at 42° C. for 1 hour and at 94° C. for 5 minutes, followed by a total of 30 cycles of 94° C. for 1 minute, 60° C. for 1 minute, and 72° C. for 1 minute, followed by reaction at 72° C. for 7 minutes. A cloning strategy for serially linking a set of cDNA fragments of four fragments is shown in
In order to increase the reconstitution efficiency of the vector, cloning was performed by locating PacI and PmeI restriction enzymes having different recognition sites and cleavage sites into a modified pBR322 vector, which is preferably a low-copy-number plasmid. The modified pBR322 vector was preferably under the control of a T7 RNA polymerase promoter and was located so that it was terminated by the hepatitis delta virus (HDV) antigenome ribozyme and T7 terminator gene used to split RNA at the terminus of the NDV genome to enable viral encapsidation and packaging. In addition, the complete genome sequence of the NDV VG/GA strain was included to ensure accurate transcription.
Thereafter, as shown in Table 2 (indicating the primers used for constructing the LVP-K1 vector of the present invention) and Table 3 (indicating the primers used for constructing the LVP-K2 vector of the present invention) below, the genes were divided into four fragments and a transgene cassette was introduced between the NP gene and the P gene and a transgene cassette was introduced between the P gene and the M gene using a cloning strategy to successively link the cDNA fragment sets.
RNA-dependent RNA polymerase initiates transcription in a sequential manner by the stop-start mechanism IGS (GE-IG-GS) between genes. In GS, the transcriptional reinitiation is not complete, so the level of transcription of mRNA located at the 3′ terminus is high. Accordingly, the higher the 3′ terminus, the higher the mRNA transcription level, and the level decreases as it goes towards the 5′ terminus. Accordingly, a new foreign gene insertion between the NP gene and the P gene results in a higher level of mRNA transcription and foreign protein translation than between the P gene and the M gene and between the HN gene and the L gene, and thus the gene insertion between NP-P is more preferable.
The four cDNA fragments had the same nucleotide sequence at the end of 15 bp, and the transgene cassette consisted of an IGS (GE-IG-GS) sequence (SEQ ID NO: 5) and a multiple cloning site (MCS). LVP-K1 (SEQ ID NO: 1) and LVP-K2 vectors for foreign gene insertion were constructed by inserting them between the NP gene and the P gene (LVP-K1) and between the P gene and the M gene (LVP-K2) using an overlap cloning method.
A recombinant Newcastle disease virus containing the PTEN protein gene in Newcastle disease virus (NDV) virus cDNA was constructed.
The LVP-K1 (NP-MCS-P) vector and the LVP-K2 (P-MCS-M) vector were inserted between the NP and P genes (LVP-K1-PTEN vector) and between the P gene and M gene (LVP-K2-PTEN vector) of the NDV cDNA backbone prepared after obtaining the required PTEN-containing DNA fragment with Fse I restriction enzyme for LVP-K1 (NP-MCS-P) vector and Not I restriction enzyme for the LVP-K2 (P-MCS-M) vector. Afterward, it was identified that the completed plasmid was 100% identical through whole gene sequencing.
Individual clones (NP, P, L) of the NDV transcriptase complex were cloned into pBR322 vector and used as helper plasmids (pBR322-NP, pBR322-P, pBR322-L). On the previous day, HEp-2 cells were prepared at 5×105 cells/well in a 6-well plate, and the modified vaccinia virus (MVA-T7) was infected at 1 MOI (multiplicity of Infection). In the cell line, each of 2.5 μg, 1.5 μg, 0.5 μg, and 5 μg of pBR322-NP, pBR322-P, pBR322-L Helper plasmids expressing proteins by the T7 promoter and LVP-K1-PTEN or LVP-K2-PTEN vectors, which are plasmids containing the PTEN protein gene were transformed by mixing them with lipofectamine 3000 (Invitrogen) at an appropriate ratio. Thereafter, the HEp-2 cell supernatant was harvested after culture at 37° C. and 5% CO2 conditions for 3 to 4 days. Then, 9-11 days old SPF embryonated eggs were inoculated into the allantoic cavity, and allantoic fluid was collected 4 days after inoculation.
Thereafter, in order to remove the vaccinia virus, the allantoic fluid diluted at 10−3 with PBS was inoculated into the allantoic cavity of 9-11 days old SPF embryonated eggs, respectively, and the allantoic fluid was collected 4 days after inoculation to conduct a virus identification experiment. For the virus identification experiment, after isolation of the allantoic fluid using a Viral RNA extraction kit (Qiagen), 5 μl of the extracted RNA and 1 μl of each of the forward and reverse primers in Table 4 below (primers necessary for identifying the construction of the recombinant NDV (LVP-K1-PTEN and LVP-K2-PTEN) of the present invention) were used for reaction at 42° C. for 1 hour and at 94° C. for 5 hours with ONE-STEP RT-PCR. Thereafter, a total of 35 cycles of reaction were carried out at 94° C. for 1 minute, at 60° C. for 1 minute, and at 72° C. for 1 minute, and then at 72° C. for 7 minutes for identification. The results are shown in
As shown in
Table 5 (primers for inserting the PTEN gene into the LVP-K1 vector) and Table 6 (primers for inserting the PTEN gene into the LVP-K2 vector) show primers for inserting the PTEN gene into the vector.
Vero76 cells were cultured at 3×105 cells/ml, and then inoculated with the recombinant virus 0.05 MOI (multiplicity of Infection) on the next day to obtain the highest titer of the virus supernatant after 2 days. Thereafter, the virus supernatant was centrifuged at 5,000 g at 4° C. for 10 minutes to remove floating materials, and the supernatant was collected. The collected supernatant was ultracentrifuged at 32,000 rpm at 4° C. for 3 hours to concentrate the recombinant virus, and after removing the supernatant, the collected supernatant was resuspended in THE buffer (10 mM Tris-HCl, 20 mM NaCl, 1 mM EDTA). The concentrated virus was subjected to ultracentrifugation at 32,000 rpm and 4° C. for 2 hours using a 30 to 60% sucrose gradient method. Recombinant virus was obtained at 40-50%. Finally, the obtained recombinant virus was subjected to ultracentrifugation once more at 32,000 rpm at 4° C. for 2 hours to remove sucrose to purify recombinant Newcastle disease virus LVP-K1-PTEN (SEQ ID NO: 2) and LVP-K2-PTEN.
In order to identify the cancer cell killing effect, T98G (CRL-1690™, ATCC) brain tumor cells were used. T98G cells were cultured as follows. T98G cells were cultured using the minimum essential medium (MEM, Gibco, USA) containing penicillin-streptomycin (Gibco, USA) and 10% FBS for cell culture, in a 37° C. incubator (5%, CO2) using a 175 T flask. When the cells grow to form a 70-80% monolayer in the flask, the cells were maintained through subculture from 1:4 to 1:6. Seeding density was about 2 to 4×104 cells/ml. Vero cells (Vero 76 KCLB No. 21587) were cultured using a Minimum essential medium (MEM, Gibco, USA) containing penicillin-streptomycin (Gibco, USA) and 10% FBS for cell culture, and a 175 T flask. When the cells grow to form a monolayer of 70 to 80% or more, subculture was proceeded and maintained. The split ratio of Vero76 cells may be up to 1:8 and the seeding density is 1×104 cells/ml.
Comparative experiments on the proliferation of LVP-K1 (SEQ ID NO: 1) virus and LVP-K1-PTEN (SEQ ID NO: 2) virus were performed using Vero cells prepared in Example 5. In general, viruses into which a foreign gene is inserted often do not proliferate well. The proliferation of the virus into which the PTEN gene was inserted was compared and identified. After removing the culture medium of Vero 76 cell (175 T flask) that has formed a monolayer of 70 to 80% or more, 10 ml of serum-free MEM medium was added to the flask and gently shake to wash the cells. After repeating this process 2 to 3 times, 5 ml (1 MOI) of LVP-K1 (SEQ ID NO: 1) virus and LVP-K1-PTEN (SEQ ID NO: 2) virus was added to the flask in advance and shook at intervals of 10 minutes in a 37° C. incubator to sensitize the virus for 1 hour. After removing the virus solution, 10 ml of serum-free MEM was put into the flask to remove the remaining virus solution, and then again, 50 ml of MEM supplemented with 5% FBS was added, and 1 ml of culture medium was collected from each flask every 4 hours, and the virus titer was measured by the TCID50 measurement method.
In order to identify that recombinant Newcastle disease viruses LVP-K1-PTEN and LVP-K2-PTEN cultured in Vero cells are maintained without PTEN gene loss during culture in Vero cells, the viruses, which have been subcultured for seven generations, were used to identify genes by RT-PCR method using the primers in Table 4 above for PTEN gene amplification. The gene obtained through electrophoresis was identified to be a PTEN gene through the sequencing method.
Pre-prepared T98G cells were inoculated with LVP-K1 (SEQ ID NO: 1) virus and LVP-K1-PTEN (SEQ ID NO: 2) virus at 1 MOI, and one flask was cultured for 6 hours without inoculating any virus. After the culture was completed, RNA was obtained from all flasks using the AccuPrep® Universal RNA extraction kit (Bioneer), and then primers and probes of Table 7 below (primers and probes for indicating the relative transcriptional amounts of PTEN mRNA in LVP-K1-PTEN and LVP-K1 virus-infected T98G cells of the present invention) were added and real-time PCR was performed. The expression level of mRNA was identified by measuring the CT value for each sample. The real time qPCR conditions were as follows. After reacting at 42° C. for 10 minutes and at 95° C. for 2 minutes, a total of 40 cycles of reaction of fluorescence level measurement were performed for 10 seconds at 95° C. and 1 minute at 59.8° C. A CFX Connect™ Real-Time System (cat. No. 1855201 Bio-Rad, USA) was used.
From the results shown in Table 3, it was understood that mRNA expression in the LVP-K1-PTEN virus-inoculated sample having the PTEN gene was higher than in the LVP-K1 virus-inoculated and non-virus-inoculated T98G cell line (control group).
Three types of viruses were inoculated into pre-prepared T98G cells, and the expression of PTEN protein in non-inoculated T98G cells was compared. There are three types of viruses: LVP-K1-PTEN (PTEN gene inserted between NP and P genes, SEQ ID NO: 2), LVP-K2-PTEN (PTEN gene inserted between P and M genes), and LVP-K1 (SEQ ID NO: 1) virus without PTEN gene inserted. Western blotting was conducted because it was determined that the amount of protein expression could be compared through an experiment in which the quantitative protein amount could not be accurately evaluated or a relative evaluation. Three types of viruses whose titer was measured in the same manner as in Example 6 or Example 8 were prepared in the T98G cell line that formed a monolayer of 80% or more, and inoculated at an MOI of 1, and then 9 hours later, the entire culture medium was frozen and thawed 3 times or more. After repeating the same, a sample was collected, the protein was separated through a conventional SDS-PAGE (Gel concentration of 12%), and the protein was transferred to a PVDF membrane (25 A, 15 minutes) using a transblottor (Thermo fisher). PVDF membrane was blocked with 1% BSA solution and then performed according to a conventional western blotting method. A PTEN monoclonal antibody was used as the primary antibody specifically binding to PTEN (Cat No. ab32119, Abcam). Anti-rabbit IgG goat horse peroxidase conjugate antibody (Invitrogen) was used as the secondary antibody to identify the protein-specific developmental reaction, and the antibody concentration was used as recommended by the manufacturer. After the secondary antibody reaction, an appropriate amount of ECL (enhanced chemiluminescence, Bio-Rad) solution was added as a color developer, and when the color developed, the protein was identified using ChemiDoc™ MP Imaging System (Bio-Rad).
As shown in the results of
In order to identify the apoptosis effect of brain tumor cells T98G cells caused by LVP-K1-PTEN virus infection, an MTT assay experiment was performed. The MTT assay method was performed according to the conventional MTT assay method, and detailed experimental procedures are described. In a 96-well plate, 1×104 T98G cells per well were cultured for 24 hours using minimum essential medium (MEM, Gibco, USA) containing penicillin-streptomycin (Gibco, USA) and 10% FBS for cell culture in a 37° C. incubator (5% CO2), and LVP-K1-PTEN virus and LVP-K1 virus were infected to be made at 0.1, 1, 2.5 and 5 MOIs. For the reliability of the results, 4 wells of the same condition were prepared to proceed. After infection, the cells were cultured in a 37° C. incubator (5% CO2), and 96 hours after infection, 20 μl of MTT solution (CellTiter 96® AQueous One solution Cell Proliferation Assay, Bio-Rad, USA) was added to each well and the cells were cultured for 1 hour in an incubator (5% CO2). Cell death was measured by measuring the absorbance of light having a wavelength of 490 nm using an iMark Microplate Reader (Bio-Rad, USA).
As a result, as shown in
After inoculation of the virus into the T98G cell culture medium, microscopic observation was performed to observe the cytopathic effect. T98G cells were cultured in a 6-well plate to form a monolayer of 90% or more, and then each virus was inoculated at 1 MOI to observe the T98G cytopathic effect under a microscope. Cytopathic effect (CPE) was observed 3 hours earlier in LVP-K1-PTEN virus-inoculated cells with PTEN gene than LVP-K1 virus-inoculated cells without PTEN gene. As a result of observation up to 18 hours thereafter, a faster cytopathic effect was observed in LVP-K1-PTEN virus-inoculated cells (see
The cancer tissue growth inhibitory effect of LVP-K1-PTEN virus was measured using a xenograft model. 6×107 cells of cultured T98G cells were dissolved in 100 ul MEM and were mixed with 100 ul Matrigel (Corning) and inoculated into the left shoulder region of mice. For the mice used, 20 SPF female BALB/c nude mice weighing about 14 to 19 g were purchased from SLC (Japan) and randomly divided into 4 mice per group for experiments. Each experimental group consisted of two groups in which LVP-K1-PTEN virus introduced with the PTEN gene was injected directly into the tail vein and cancer tissue, two groups in which LVP-K1 virus without PTEN gene introduced was injected directly into the tail vein and cancer tissue, and a PBS inoculation group (control group). The concentration of the inoculated virus was all 108.0 TCID50/ml, and 100 ul each was inoculated. Virus inoculation was performed twice at an interval of 2 days from when the cancer tissue size reached an average of 120 to 150 mm3 on the 7th day after T98G cell inoculation, and cancer tissue changes were observed every day for 7 days. The size of the cancer tissue was calculated using the formula 4π/3×(smallest diameter/2)2×(largest diameter/2)2.
Though table 8 (LVP-K1-PTEN and LVP-K1 viruses of the present invention were injected directly into the tail vein or cancer in xenograft to reduce cancer size), and
In conclusion, it was identified that cancer cell proliferation inhibitory effect can be obtained through the production of a novel cancer treatment viral vector using the NDV virus and a virus using the same that does not cause an immune response and does not generate antibodies in mammals. There are many NDV viruses that disappear from normal cells without reaching cancer cells due to innate immunity immediately after infection, which is a major disadvantage and advantage of NDV virus. In addition, due to the dilution effect of intravenous injection, it is a very important task to develop a recombinant NDV virus, rather than a simple recombinant virus that has been used already, that is expressed as a protein in cancer cells to increase efficacy such as cancer cell proliferation inhibitory effect, apoptosis induction, and immune response induction by inserting various genes. Further research will be needed in the future for further development.
The present invention demonstrates that there may be an oncolytic function to some extent in cancer cells in which the normal PTEN protein function is lost due to PTEN gene mutation in brain tumors and the NDV recombinant virus capable of additionally introducing a PTEN gene and passing a brain blood barrier along the nervous system is produced and the effect thereof is improved.
The disclosed embodiments should be considered in an illustrative in all aspects rather than a restrictive perspective. The scope of the present invention is defined by the following claims rather than by the preceding description. It should be interpreted that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.
Depository Institution: Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Biotechnology and Bioscience
Accession No.: KCTC14496BP
Deposit Date: Mar. 12, 2021
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0038880 | Mar 2021 | KR | national |
This application is a National Stage of International Application No. PCT/KR2021/006924, filed Jun. 3, 2021, claiming priority to Korean Patent Application No. 10-2021-0038880, filed Mar. 25, 2021, the entire disclosures of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/006924 | 6/3/2021 | WO |
