WO2017068614A1 - Composition de thérapie génique - Google Patents

Composition de thérapie génique Download PDF

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WO2017068614A1
WO2017068614A1 PCT/JP2015/005342 JP2015005342W WO2017068614A1 WO 2017068614 A1 WO2017068614 A1 WO 2017068614A1 JP 2015005342 W JP2015005342 W JP 2015005342W WO 2017068614 A1 WO2017068614 A1 WO 2017068614A1
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gene
apoptosis
composition
fus
inducing
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Japanese (ja)
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徹幸 平畑
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Priority to MYPI2018701967A priority Critical patent/MY188689A/en
Priority to PCT/JP2015/005342 priority patent/WO2017068614A1/fr
Priority to SG11201803940PA priority patent/SG11201803940PA/en
Priority to JP2017546280A priority patent/JP6837984B2/ja
Publication of WO2017068614A1 publication Critical patent/WO2017068614A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a composition for gene therapy, and in particular, a composition for gene therapy for promoting apoptosis activity of cancer cells by introducing a specific apoptosis-inducing gene into the cancer cells, and the composition for gene therapy as cancer.
  • the present invention relates to a liposome vector for transfer to a cell and a method for forming the same.
  • Each of these therapies is merely a symptomatic treatment for tumors in the visible range caused by genetic alterations, and there is a problem that it is difficult to completely cure cancer.
  • Gene therapy is a therapy that treats cancer by correcting the defects of cells that have malfunctioned due to having an abnormal gene by administering the gene or a cell into which the gene has been introduced and reducing the gene abnormality. Therapies using various genes have been developed.
  • a composition for gene therapy comprising mesenchymal stem cells derived from umbilical cord blood for diagnosis, prevention, treatment, or monitoring of treatment progress of brain tumors
  • Techniques relating to these compositions or kits are disclosed.
  • brain tumors can be treated by inducing and promoting apoptosis of cancer cells using an anti-tumor gene comprising an apoptosis-inducing factor gene using a cytokine or an interleukin gene.
  • this technique can surely eliminate the brain tumor (cancer), it is possible to select a composition according to the patient's constitution, type of cancer, progress, etc., that is, from the viewpoint of customization. It was not enough. In addition, a vector for accurately introducing the anti-tumor gene with cancer cells is required, but this point is also insufficient.
  • the present invention provides a gene therapy composition that promotes apoptosis activity of cancer cells by introducing an apoptosis-inducing gene comprising a combination of a plurality of genes into cancer cells. And a liposome vector for introducing the gene therapy composition into cancer cells and a method for forming the same.
  • a gene therapy composition according to the present invention is a gene therapy composition for preventing and / or treating cancer, wherein the antitumor gene comprises an apoptosis-inducing gene, wherein the apoptosis-inducing gene is used.
  • the gene is composed of a p53 gene, a FUS-1 gene, a TRAIL gene, and an IL-24 gene. Further, the apoptosis-inducing gene is configured such that the contents of the p53 gene, the FUS-1 gene, the TRAIL gene, and the IL-24 gene are equal.
  • the apoptosis-inducing gene has a configuration in which the p53 gene, the TRAIL gene, and the IL-24 gene are double the amount of the FUS-1 gene in order to activate apoptosis induction.
  • the p53 gene and the FUS-1 gene are the TRAIL gene and the IL-24 gene. The structure is double the amount.
  • the apoptosis-inducing gene has a configuration in which the p53 gene, the FUS-1 gene and the TRAIL gene are double the amount of the IL-24 gene in order to activate non-p53-dependent apoptosis induction.
  • the apoptosis-inducing gene has a structure in which the p53 gene is double the amount of the FUS-1, TRAIL, and IL-24 genes in order to activate p53-dependent apoptosis induction.
  • the FUS-1 gene is 2 to 5 times the amount of the p53 gene, the TRAIL gene and the IL-24 gene. It is a configuration.
  • the apoptosis-inducing gene has a constitution in which the IL-24 gene is double the amount of the p53 gene, the FUS-1 gene and the TRAIL gene in order to obtain an antitumor effect against melanoma.
  • the apoptosis-inducing gene has a constitution in which the FUS-1 gene and the IL-24 gene are double the amount of the p53 gene and the TRAIL gene in order to obtain an antitumor effect against lung cancer and / or breast cancer.
  • the apoptosis-inducing gene has a structure in which the TRAIL gene and the IL-24 gene are double the amount of the p53 gene and the FUS-1 gene in order to increase the anti-angiogenic action and the sensitivity to radiation therapy. .
  • the apoptosis-inducing gene has a structure in which the p53 gene and the TRAIL gene are double the amount of the FUS-1 gene and the IL-24 gene in order to reduce the antitumor effect and side effects in combination with chemotherapy. It is.
  • the gene therapy composition further comprises a p16 gene and / or a p21 gene, and the content of each gene is equal.
  • the gene therapy composition is encapsulated by a gene therapy liposome vector comprising cationic lipid and helper lipid liposomes targeting cancer cells.
  • the liposome comprises a monolayer membrane having an internal cavity, and the membrane comprises a capsule-like vector to which a ligand is attached, and comprises a monolayer thin film containing dioleoylphosphatidylethanolamine and dimethylaminoethanecarbamoylcholesterol It is the structure which consists of these compounds.
  • the gene therapy composition further comprises a gene obtained by plasmiding a proto-oncogene and a cytokine gene.
  • the cytokine gene is composed of an IL-12 gene or an IL-18 gene.
  • the method for forming a liposome for gene therapy comprises a mixing step of mixing dioleoylphosphatidylethanolamine and dimethylaminoethanecarbamoylcholesterol in chloroform to form a lipid solution, and drying the generated solution.
  • a film forming step for producing a thin film a vacuum step for maintaining the produced thin film in vacuum, a resuspension step for resuspending the thin film by adding water, dextrose, or vortex, and the suspended suspension.
  • a culture step of culturing a suspension at 4 ° C. an ultrasonic step of performing ultrasonic irradiation treatment to form liposomes from the suspension or the cultured suspension, and a polycarbonate having the same size as the size of the liposomes to be produced And a forming step of extruding liposomes with a filter.
  • each gene of p53, FUS-1, TRAIL, and IL-24 is mixed in an equal amount, so that it becomes possible to acclimate to the body without giving a sudden impact to the patient, and in the subsequent treatment It is possible to determine the amount of each gene to be administered.
  • each gene of p53, TRAIL, and IL-24 is configured to contain about twice the amount of the FUS-1 gene, so that it is possible to activate apoptosis induction.
  • p53 and FUS-1 genes are contained as much as TRAIL and IL-24 genes as apoptosis-inducing genes, MDM2 activation is suppressed and p53-dependent apoptosis induction is activated. Is possible. 5). Since the p53, FUS-1 and TRAIL genes are contained as much as the amount of IL-24 gene as the apoptosis-inducing gene, it is possible to activate apoptosis induction independent of the p53 gene.
  • the p53 gene is contained in an amount approximately double that of the FUS-1, TRAIL, and IL-24 genes, so that it is possible to activate apoptosis induction depending on the p53 gene. 7).
  • the FUS-1 gene is contained in 2 to 5 times the amount of p53, TRAIL, and IL-24 genes, so that it is possible to suppress the growth of cancer cells through the MAPK pathway. Become.
  • an IL-24 gene is contained in an amount twice that of p53, FUS-1 and TRAIL genes, so that an antitumor effect against melanoma can be obtained.
  • the FUS-1 and IL-24 genes are contained in an amount twice that of the p53 and TRAIL genes, so that an antitumor effect against lung cancer and / or breast cancer can be obtained.
  • the p53 and TRAIL genes are contained in an amount about double that of the FUS-1 and IL-24 genes, so that it is possible to obtain an anti-tumor effect in combination with chemotherapy and have side effects. Can be reduced.
  • the TRAIL and IL-24 genes are contained in double the amount of the p53 and FUS-1 genes, so that it is possible to obtain an anti-angiogenic action and an effect of increasing sensitivity to radiation therapy. Become.
  • composition for gene therapy further contains the p16 gene and the p21 gene and each gene is contained in an equal amount, activation of apoptosis induction in all cancer cells can be achieved. . 13. Since the gene therapy composition comprising various genes is encapsulated by the liposome vector for gene therapy comprising liposomes, the stability of the gene therapy composition can be ensured and the gene therapy composition can be reliably applied to the target tumor cells. It becomes possible to deliver.
  • the liposome is composed of a monolayer membrane having an internal cavity, it is possible to construct a vector in which the composition for gene therapy is stored. 15. Since the composition for gene therapy further comprises a gene obtained by plasmiding a proto-oncogene and a cytokine gene, it is possible to suppress the growth of tumor cells and induce apoptosis.
  • the liposome is formed from the suspension by the ultrasonic irradiation treatment, the liposome having a size of about 200 nm can be formed.
  • FIG. 1 is a list showing combinations of apoptosis-inducing genes.
  • the composition for gene therapy according to the present invention is a composition for preventing and / or treating cancer mainly by the action of an anti-tumor gene. By using this composition via a vector, it can be applied to a tumor (cancer cell). A composition for treatment by direct introduction and killing cancer cells.
  • the anti-tumor gene is composed of an apoptosis-inducing gene having apoptosis characteristics. That is, by introducing a gene having apoptosis-inducing properties into cancer cells, the cancer cells are extinguished by inducing spontaneous death (self-death) of the cancer cells. That is, a composition for gene therapy comprising a composition for preventing and / or treating cancer can be provided.
  • the apoptosis-inducing gene has a composition comprising all four types of genes: p53 gene, FUS-1 gene, TRAIL gene, and IL-24. These induced genes are all tumor suppressor genes, and it has been experimentally confirmed that they have a high antitumor effect in cancer cell killing treatment using these genes.
  • the present invention seeks to elucidate and pursue that the composition always contains all of these four types of genes, and that the composition has different properties by changing the content ratio (weight percent).
  • the gene of p53 is a typical tumor suppressor gene, and is a responsible gene for Li-Fraumeni syndrome, a hereditary disease that is a risk of developing malignant tumors in various organs in childhood and adulthood. It is. Also in experiments, p53-deficient mice in which the p53 gene has been lost are experimentally proven to be an important factor for cancer suppression because of the occurrence of tumors at a high frequency at an early stage after birth. p53 is induced by DNA damage and various stresses, and functions as a transcriptional activator in the cell nucleus.
  • P53 is activated in cells that have been stimulated by DNA damage, and controls cell cycle checkpoints to stop cell division and promote DNA repair during that time, thereby suppressing DNA mutation. In this process, if the cells cannot be repaired, gene alteration is prevented by inducing and eliminating apoptosis (cell death). It has been clarified from experiments that p53 is unstable when its function is lost. In addition, experiments have shown that expression of oncogenes in normal cells leads to p53-dependent apoptosis and induction of cell senescence. By eliminating cells with activated oncogenes, canceration can be prevented. It is clear that it is suppressed.
  • the FUS-1 gene is a tumor suppressor gene present in the human chromosome 3p21.3 region, and the disappearance and mutation of FUS-1 alleles (individual genes that define alleles) are observed early in many cancers. In particular, disappearance or phenomenon of FUS-1 protein is observed in lung cancer, lung tumor, and breast cancer. FUS-1 induces cancer cells to apoptosis by activating mitochondria-dependent Apaf-1-related pathways and suppressing the function of tyrosine kinase proteins including EGFR, PDGFR, AKT, c-Abl, and c-Kit It has become clear.
  • FUS-1 is activated by apoptosis stimulation, stress, etc., and activates apoptosis of endogenous mitochondria.
  • activation of FUS-1 has the property of blocking the suppression of p53 activity by the MDM2 gene and enhancing p53-dependent apoptosis.
  • the gene of FUS-1 is a gene having a function of suppressing cell proliferation due to MDM2 expression and EGFR mutation. Suppresses the growth of cancer cells through the MAPK pathway.
  • the FUS-1 gene is known to have an antitumor effect on lung cancer and breast cancer.
  • the TRAIL gene is a gene that has the property of leading cells to apoptosis via the death receptor.
  • the TRAIL gene can be used as a gene specialized in cancer treatment because it selectively induces various tumor cells and abnormal cells to apoptosis and does not act on normal cells.
  • the TRAIL gene is a homotrimeric ligand that interacts with members of the TNF receptor family.
  • IL-24 interleukin-24 gene
  • IL-24 does not act on normal cells and has the effect of enhancing the sensitivity of cancer cells to radiation.
  • IL-24 exhibits an antitumor effect against melanoma, lung cancer and breast cancer. It is also known to have an angiogenesis inhibitory effect.
  • the gene therapy composition according to the present invention is based on information such as gene abnormality and diagnosis name obtained by gene diagnosis, presence / absence of metastasis, etc., p53 gene, FUS-1 gene, TRAIL gene and IL- Allocation is determined by specifying the content of four genes of 24 genes.
  • the p53 gene, the FUS-1 gene, the TRAIL gene, and the IL-24 gene constituting the apoptosis-inducing gene are contained in equal amounts.
  • the p53 gene, the FUS-1 gene, the TRAIL gene, and the IL-24 gene constituting the apoptosis-inducing gene are contained in equal amounts.
  • by adjusting the dose it is possible to enhance the effect of the gene therapy composition in a balanced manner.
  • by monitoring the expression of various genes such as MDM2 it is possible to determine the distribution of the above genes next.
  • the apoptosis-inducing gene may be configured to contain a p53 gene, a TRAIL gene, and an IL-24 gene in double the amount of the FUS-1 gene.
  • a gene therapy composition containing equal amounts of each of the above genes is used, if a p53 mutation is observed, it is necessary to administer an increased amount of p53 and promote p53-independent apoptosis induction.
  • p53 and p53-independent activation of apoptosis can be achieved by including the p53 gene, TRAIL gene and IL-24 gene in double amounts of the FUS-1 gene.
  • An effective gene therapy composition can be provided.
  • the apoptosis-inducing gene of the gene therapy composition contains a p53 gene and a FUS-1 gene in double amounts of the TRAIL gene and the IL-24 gene. It is said.
  • a gene therapy composition containing equal amounts of each of the above genes if overexpression of MDM2, AKT1 (active kinase), or EGFR is observed, it is necessary to suppress cell proliferation or induce apoptosis Will occur.
  • the FUS-1 gene twice as much as the TRAIL gene and the IL-24 gene, it is possible to suppress the expression of MDM2 and activate the p53-dependent caspase pathway and induce cancer cells to apoptosis It becomes possible to do.
  • FUS-1 makes it possible to suppress cell growth due to EGFR abnormalities, and thus a tumor growth inhibitory effect can be expected.
  • overexpression of MDM2, AKT1, and EGFR affects the growth of cancer cells by inhibiting the induction of apoptosis by p53, it is possible to activate p53-dependent apoptosis induction by increasing the amount of p53. Thus, it is possible to provide a more effective composition for gene therapy.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention can be configured to contain a p53 gene, a FUS-1 gene, and a TRAIL gene in double the amount of the IL-24 gene. is there.
  • BCL2 When overexpression of BCL2 is observed when the gene therapy composition is used, it is necessary to suppress anti-apoptotic activity. Therefore, by containing double amounts of the genes for p53 and FUS-1, cancer cells can be induced to apoptosis as described above, and a tumor growth inhibitory effect can be expected.
  • a double amount of the TRAIL gene non-p53-dependent activation of apoptosis induction can be achieved, and a more effective gene therapy composition can be provided.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention can be configured such that the p53 gene contains double the amount of the FUS-1, TRAIL, and IL-24 genes. .
  • the p53 gene contains double the amount of the FUS-1, TRAIL, and IL-24 genes.
  • methylation of the APC gene or mutation of BRCA-1 or BRCA-2 that is a tumor suppressor gene is observed when the gene therapy composition is used, it is necessary to enhance p53-dependent apoptotic activity. Therefore, p53-dependent activation of apoptosis induction can be achieved by containing a p53 gene in a double amount compared to the others. In this case, it is desirable that the p53 gene is contained twice or more in comparison with other genes.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention is configured to contain FUS-1 gene twice to five times the amount of p53 gene, TRAIL gene and IL-24 gene. Is possible.
  • the gene therapy composition is used, overexpression of any of RAS (H-RAS, K-RAS, N-RAS), MAP2K5, MAP3K10, and RAF (B-RAF, A-RAF) is observed. Then, it is necessary to suppress the growth of cancer cells through the MAPK pathway. For this reason, it is configured to contain 2 to 5 times the amount of FUS-1 depending on the number of overexpressed. In the present example, the amount is increased by 2 times by 1 overexpression, 3 times by 3 overexpression, 4 times by 5 overexpression, 5 times by 5 or more overexpression. Thereby, the tumor growth inhibitory effect can be expected.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention can be configured to contain IL-24 gene in double the amount of p53 gene, FUS-1 gene and TRAIL gene. .
  • IL-24 gene when VEGF-A is overexpressed when the above gene therapy composition is used, an anti-angiogenic action can be expected. Therefore, the composition of IL-24 is doubled. ing. This makes it possible to prevent the growth and metastasis of cancer tissue due to the neovascularization.
  • the IL-24 gene exerts an antitumor effect against melanoma, it can be expected to induce apoptosis of cancer cells and suppress tumor growth by containing double the IL-24 gene.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention is configured to contain FUS-1 gene and IL-24 gene in double amounts of p53 gene and TRAIL gene.
  • the FUS-1 and IL-24 genes can be expected to have an antitumor effect on lung cancer and breast cancer. For this reason, in this example, when the clinical diagnosis name is lung cancer and / or breast cancer, the FUS-1 and IL-24 genes are contained in double amounts. As a result, cancer cell apoptosis induction and tumor growth-suppressing effects can be expected particularly for lung cancer and breast cancer.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention can be configured to contain TRAIL gene and IL-24 gene in double amounts of p53 gene and FUS-1 gene.
  • this example is configured to contain double amounts of TRAIL and IL-24 genes.
  • the apoptosis-inducing gene of the gene therapy composition according to the present invention can be configured to contain a p53 gene and a TRAIL gene in double amounts of the FUS-1 gene and the IL-24 gene. . With this configuration, it is possible to expect an antitumor effect in combination with chemotherapy, and to reduce side effects.
  • the gene therapy composition according to the present invention may further comprise a p16 gene and a ⁇ 21 gene.
  • the gene for p16 is a tumor suppressor gene and plays an important role in the regulation of the cell cycle. Mutations in the p16 gene increase the risk of developing various cancers, particularly melanoma.
  • the p21 gene is a gene that inhibits the progression of the cell cycle by inhibiting a cyclin-dependent kinase that affects the rotation of the cell cycle. By containing these in the same amount as other genes, apoptosis induction of tumor cells and tumor growth inhibitory effect can be expected.
  • composition for gene therapy according to the present invention can be configured to be encapsulated with a liposome vector for gene therapy.
  • the gene therapy composition of the present invention is encapsulated by liposomes, which are non-viral vectors, and is directly introduced into target cancer cells.
  • the liposome vector for gene therapy is composed of a liposome of a cationic lipid and a helper lipid, and is composed of a capsule-shaped vector composed of a monolayer thin film compound having an internal cavity.
  • the liposome has a diameter of 70 nm to less than 100 nm, and the inner space has an average of 50 nm.
  • a gene such as a plasmid or a composition for gene therapy is stored.
  • Ligand is attached to the liposome monolayer. Thereby, binding to a specific receptor (cancer cell) can be expected, and the composition for gene therapy can be more effectively introduced into the target cancer cell.
  • this liposome contains at least dioleoylphosphatidylethanolamine and dimethylaminoethanecarbamoylcholesterol, and is formed of a cation as a lipid for assisting membrane fusion by these.
  • the stability of the gene therapy composition can be ensured, and the gene therapy composition can be protected from any disorder outside the cell, which is surely the target cancer. It becomes possible to introduce therapeutic DNA or RNA (a composition for gene therapy) into cells.
  • composition for gene therapy according to the present invention may further comprise a gene obtained by converting a proto-oncogene into a plasmid.
  • a gene obtained by converting a proto-oncogene obtained by genetic diagnosis into a plasmid By converting the proto-oncogene obtained by genetic diagnosis into a plasmid, the plasmid is incorporated into the nucleus of the cell, and a protein is produced as an endogenous antigen from DNA through a transcription / translation process. Thereafter, antigens are presented by MHC class 1 molecules, cancer cells are rejected, and killer T cells cause cell damage.
  • a cell having a protein as an endogenous antigen undergoes apoptosis, and the endogenous antigen is released to the outside of the cell, so that it is phagocytosed by a phagocytic cell such as a dendritic cell.
  • a phagocytic cell such as a dendritic cell.
  • cytokine gene it is possible to further contain a cytokine gene as a gene therapy composition.
  • the cytokine gene contains an IL-12 gene or an IL-18 gene.
  • IL-12 exhibits actions such as promoting cell proliferation and inducing cytotoxic activity against T cells and natural killer cells (NK cells) that act as a major factor of innate immunity. Since it has such a role of cellular immune function, IL-12 can be expected to be effective in infection protection, anticancer therapy, improvement of immune deficiency, and the like.
  • IL-18 is a gene related to mass production of INF- ⁇ that responds to toxic stimuli, and also stimulates macrophages to phagocytose bacteria. Because of having such a role, IL-18 can be expected to be effective in infection protection, anticancer therapy and the like.
  • Fas ligand is a cytokine that induces apoptosis by transducing a signal by trimerizing a Fas receptor that penetrates the membrane of a target cell, and is a cell death-inducing THF receptor family molecule. Induces apoptosis in expressing tumor cells. That is, a reduction in cancer cells and a tumor growth inhibitory effect can be expected.
  • NK cells selectively remove abnormal cells that escape immune surveillance by T cells due to decreased expression of MHC class 1 molecules.
  • MHC class 1 molecules are not necessarily NK-sensitive, even if normal NK cells are cultured and introduced into cancer cells, in terms of antitumor effects , NK cells may not be activated.
  • surface receptors for proteins (cancer antigens and ligands) expressed in cancer cells can be expressed in NK cells by introducing proto-oncogenes into NK cells outside the body based on genetic diagnosis. Even if the cells have MHC class 1 molecules, the receptor that binds to the activated NK receptor ligand is expressed, so that the NK cells can specifically kill cancer cells and are more effective. It is possible to reduce cancer cells and suppress tumor growth.
  • the gene therapy composition according to the present invention can be further introduced together with dendritic cells.
  • Dendritic cells are a type of immune cell that functions as an antigen-presenting cell, and have a role of transmitting the antigen taken up to cells of other immune systems. When antigen is taken up, dendritic cells are activated and migrate to secondary lymphoid organs such as lymph nodes and spleen. In lymphoid organs, T cells specific for the incorporated antigen are activated. Since this activation is efficient, it can be said that dendritic cells are superior to macrophages in activating T cells.
  • a proto-oncogene obtained by genetic diagnosis is used as an antigen, and a plasmid is placed in a liposome and introduced into dendritic cells. This has the advantage that it is not necessary to remove the cancer tissue.
  • the gene therapy composition according to the present invention can be introduced together with the NK cell or the dendritic cell, and can be introduced together with both the NK cell and the dendritic cell. Furthermore, a high antitumor effect can be expected by introducing a proto-oncogene together with a plasmided gene (DNA vaccine) or cytokine gene.
  • the liposome for gene therapy consists of a mixing process, a film production process, a vacuum process, a resuspension process, a culture process, an ultrasonic process, and a formation process.
  • the mixing step is a step in which dioleoylphosphatidylethanolamine and dimethylaminoethanecarbamoylcholesterol are mixed in chloroform to form a lipid solution. This produces a lipid solution.
  • the produced lipid solution is dried by N2 gas blowing in the next film production process. Thereby, a thin film is produced
  • the thin film dried and produced in the film production process is maintained in vacuum. In this embodiment, the vacuum state is maintained for 5 hours while the opening of the container is covered.
  • the thin film maintained in vacuum is suspended in the resuspension process.
  • resuspension is performed by adding either water or dextrose or vortex to the membrane.
  • the suspended suspension is cultured in the culture process.
  • the culture is performed at 4 ° C. for 24 hours.
  • the culture time is not limited to this, and the culture time can be appropriately changed. In this embodiment, it is possible to move to the next ultrasonic step without performing this culture step.
  • ultrasonic irradiation treatment is performed to form liposomes from the suspension or the suspension cultured through the culture process.
  • the forming step in order to further control the size of the liposome, after determining the size to be produced, the liposome is extruded and formed through a polycarbonate filter having the same size as the size of the liposome to be produced.
  • temperature control is performed during the extrusion formation of liposomes. This step makes it possible to produce unilamellar liposomes.
  • the size of the generated liposome is less than 100 nm (nanometers).
  • This gene therapy liposome production method makes it possible to reliably produce liposomes as vectors for gene therapy compositions, and facilitate introduction of gene therapy compositions into target cancer cells. Became possible.

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Abstract

Le problème décrit par la présente invention est de fournir une composition de thérapie génique qui stimule l'activité apoptotique dans des cellules cancéreuses par la transfection dans lesdites cellules cancéreuses d'un gène induisant l'apoptose comprenant une combinaison d'une pluralité de gènes, un vecteur liposomique permettant de transfecter la composition de thérapie génique dans lesdites cellules cancéreuses, et un procédé de formation dudit vecteur liposomique. La solution selon l'invention porte sur une composition de thérapie génique qui est destinée à la prévention et/ou au traitement d'un cancer et sur ses gènes antitumoraux qui comprennent un gène induisant l'apoptose. Ledit gène induisant l'apoptose est une construction comprenant le gène p53, le gène FUS-1, le gène TRAIL, et le gène IL-24.
PCT/JP2015/005342 2015-10-23 2015-10-23 Composition de thérapie génique Ceased WO2017068614A1 (fr)

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Application Number Priority Date Filing Date Title
MYPI2018701967A MY188689A (en) 2015-10-23 2015-10-23 Gene therapy composition
PCT/JP2015/005342 WO2017068614A1 (fr) 2015-10-23 2015-10-23 Composition de thérapie génique
SG11201803940PA SG11201803940PA (en) 2015-10-23 2015-10-23 Gene therapy composition
JP2017546280A JP6837984B2 (ja) 2015-10-23 2015-10-23 遺伝子治療用組成物

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