WO2024112118A1 - Composition d'agent thérapeutique cellulaire hautement fonctionnel pour régénérer des muscles endommagés par une radiothérapie - Google Patents

Composition d'agent thérapeutique cellulaire hautement fonctionnel pour régénérer des muscles endommagés par une radiothérapie Download PDF

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WO2024112118A1
WO2024112118A1 PCT/KR2023/018990 KR2023018990W WO2024112118A1 WO 2024112118 A1 WO2024112118 A1 WO 2024112118A1 KR 2023018990 W KR2023018990 W KR 2023018990W WO 2024112118 A1 WO2024112118 A1 WO 2024112118A1
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gene
nucleotide
base sequence
myf6
myog
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정은재
정석
김혜정
김인걸
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고려대학교 산학협력단
서울대학교병원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • This invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under project number 1711200059 and task number 2022M3A9B6018217.
  • the research management agency for the project is the National Research Foundation of Korea, the research project name is "Next Generation Applied Omics Project”, and the research project name is “Development of an integrated meta-tissue chip to build models of respiratory infectious diseases and steatohepatitis”, hosted by Korea University, research period: 2022.04.01. ⁇ 2025.12.31.
  • This invention was made with the support of the Ministry of Health and Welfare of the Republic of Korea under Project No. 1465037194 and Project No. HI22C1323000022.
  • the research management agency for the project is the Korea Health Industry Development Institute, the research project name is "Regenerative Medicine Clinical Research Base Creation Project”, and the research project is Myeong-eun "Development of 3D printing bioink for artificial esophagus based on ECM/CNF using immunomodulatory bioactive nanoparticles to control foreign body reaction/fibrosis", Host institution is Seoul National University Hospital, Research period is 2022.07.01. ⁇ 2026.12.31.
  • the present invention can regenerate damaged muscle tissue by transfecting it with a vector co-introduced with MyoG, MyoD, and Myf6, which are genes for improving muscle differentiation.
  • MyoG, MyoD, and Myf6 are genes for improving muscle differentiation.
  • the degree of damage to the esophageal muscle layer damaged by radiation for the treatment of head and neck cancer can be measured. It relates to stem cells that can improve, prevent or treat damage and methods of producing the same.
  • Cancer is a group of diseases characterized by abnormal regulation of cell growth. There are over 100 different types of cancer, classified according to the type of cell initially affected, including bladder, breast, colon, rectal, endometrial, and kidney (renal cell carcinoma) cancers. , leukemia, small cell lung cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, thyroid cancer, skin cancer, non-Hodgkin's lymphoma, melanoma, and head and neck cancer.
  • head and neck cancer is the 6th most common cancer worldwide, with an incidence of over 640,000 cases worldwide each year.
  • Head and neck squamous cell carcinoma (HNSCC) accounts for more than 90% of all head and neck cancers, making up the majority of head and neck cancers, and occurs on mucosal surfaces throughout the anatomical region. These include tumors of the nasal cavity, paranasal sinus, oral cavity, nasopharynx, oropharynx, hypopharynx, and larynx. 35% to 45% of head and neck cancer patients ultimately die due to head and neck squamous cell carcinoma disease. In the United States alone, head and neck squamous cell carcinoma accounts for approximately 4% of all malignant tumors. This corresponds to approximately 17 of the 100,000 people newly diagnosed with head and neck squamous cell carcinoma each year.
  • Radiation therapy is the standard treatment for most head and neck cancers, including upper digestive tract cancers, and radiation therapy is generally performed on patients in combination with surgical therapy.
  • the number of surgical cases for head and neck cancer is increasing every year, and radiation therapy for head and neck cancer exposes the normal bones and soft tissues of the jaw, face, and neck to radiation, causing tissue damage known as radiation fibrosis.
  • Radiation fibrosis can occur in all soft tissues, including skin, connective tissue, muscles, nerves, and blood vessels.
  • a typical side effect that occurs after radiation therapy to the upper digestive system, including the mouth, pharynx, and esophagus is dysphagia. This is a decline in function due to tissue fibrosis of the mucosa and muscle layer of the esophagus, causing permanent discomfort and pain to the patient.
  • Radiotherapy is known to promote the death of stem cells, inflammatory response, and activation of fibroblasts in tissues through ionized radiation. As a result, excessive deposition of collagen and other extracellular matrix components occurs in tissues exposed to radiation, leaving permanent tissue scars.
  • the present inventors focused on the fact that the esophageal muscle is the most important target tissue for restoring the fundamental function of the esophagus, and developed a therapeutic agent that can ultimately treat damaged esophageal tissue with stem cells by restoring key genes in the damaged esophageal muscle layer after radiation. I tried to make it.
  • transfecting stem cells with a vector combining the transcription factor MyoG, MyoG, and Myf6 genes increased the muscle tissue regeneration effect of stem cells more than transfecting stem cells with vectors containing each gene. It was confirmed that the effect of improving esophageal fibrosis was excellent.
  • the purpose of the present invention is to provide a vector for producing stem cells for muscle tissue regeneration containing MyoG, MyoD, and Myf6 genes.
  • Another object of the present invention is to provide stem cells for muscle tissue regeneration transfected with a vector carrying MyoG, MyoD and Myf6 genes.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating dysphagia.
  • Another object of the present invention is to provide a method for producing a composition for muscle tissue regeneration.
  • Another object of the present invention is to provide a use of stem cells containing MyoG, MyoD and Myf6 genes for muscle tissue regeneration.
  • the present invention can regenerate damaged muscle tissue by transfecting it with a vector co-introduced with MyoG, MyoD, and Myf6, which are genes for improving muscle differentiation, and specifically, the degree of damage to the esophageal muscle layer damaged by radiation for the treatment of head and neck cancer. It relates to stem cells that can improve, prevent or treat damage, and methods for producing the same.
  • An example of the present invention relates to a vector for producing stem cells for muscle tissue regeneration.
  • the vector for producing stem cells for muscle tissue regeneration may include a first nucleotide containing the MyoG gene, a second nucleotide containing the MyoD gene, and a third nucleotide containing the Myf6 gene.
  • a vector may refer to a means for expressing a target gene in a host cell.
  • it may include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors.
  • Vectors that can be used as recombinant vectors include plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), phages (e.g., ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, and M13, etc.), or viruses (e.g., SV40, etc.).
  • plasmids often used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1,
  • the recombinant vector can typically be constructed as a vector for cloning or a vector for expression.
  • Expression vectors may be those commonly used in the art to express foreign proteins in plants, animals or microorganisms, but are not limited thereto and can be constructed through various methods known in the art.
  • the vector contains elements for expression of the target gene, and may include a replication origin, a promoter, an operator, a transcription termination sequence, etc., and may include a host cell. Appropriate enzymatic sites (e.g., restriction enzyme sites) for introduction into the genome and/or selection markers to confirm successful introduction into host cells and/or ribosome binding site (RBS) for translation into proteins. ), IRES (Internal Ribosome Entry Site), etc. may be additionally included.
  • the vector may further include transcriptional control sequences (eg, enhancers, etc.) other than the promoter.
  • the recombinant vector can be constructed using prokaryotic cells or eukaryotic cells as hosts.
  • a strong promoter capable of advancing transcription e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.
  • a ribosome binding site for initiation of translation e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.
  • a transcription/translation termination sequence e.g., pL ⁇ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.
  • the origin of replication operating in the eukaryotic cell included in the vector includes the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and the BBV origin of replication. It is not limited.
  • promoters derived from the genome of mammalian cells e.g., metallothioneine promoter
  • promoters derived from mammalian viruses e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV
  • adenovirus late promoter vaccinia virus 7.5K promoter
  • SV40 promoter SV40 promoter
  • the cytomegalovirus promoter and the tk promoter of HSV can be used and typically have a polyadenylation sequence as the transcription termination sequence.
  • a vector may refer to an expression vector for gene therapy.
  • gene therapy refers to a method of inserting a normal gene into a cell with a genetic abnormality or providing a new function to normalize its function in order to treat various diseases caused by genetic abnormalities. can do.
  • tissue may refer to a part of one or more organs selected from the group consisting of the esophagus, heart, lung, and liver.
  • tissue may be a part of the esophagus, but is not limited thereto.
  • the first nucleotide containing the MyoG gene may include the base sequence of SEQ ID NO: 1 or a base sequence substantially identical to the base sequence of SEQ ID NO: 1.
  • Substantial identity is determined by aligning each nucleotide sequence with any other nucleotide sequence to correspond as much as possible, and analyzing the sequence, so that any other nucleotide sequence is at least 70%, 80%, 90% or more identical to each nucleotide sequence. This may mean having sequence homology of 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, or 98% or more.
  • the nucleotide sequence having substantial identity to the nucleotide sequence of SEQ ID NO: 1 is obtained by aligning the nucleotide sequence of SEQ ID NO: 1 and any other nucleotide sequence to correspond as much as possible, analyzing the sequence, and The base sequence of SEQ ID NO: 1 and at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% This may mean having more than one sequence homology.
  • the second nucleotide containing the MyoD gene may include the base sequence of SEQ ID NO: 2 or a base sequence substantially identical thereto.
  • the nucleotide sequence having substantial identity to the nucleotide sequence of SEQ ID NO: 2 is obtained by aligning the nucleotide sequence of SEQ ID NO: 2 and any other nucleotide sequence to correspond as much as possible, analyzing the sequence, and The base sequence of this SEQ ID NO: 2 and at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% This may mean having more than one sequence homology.
  • the third nucleotide containing the Myf6 gene may include the base sequence of SEQ ID NO: 3 or a base sequence substantially identical thereto.
  • the nucleotide sequence having substantial identity to the nucleotide sequence of SEQ ID NO: 3 is obtained by aligning the nucleotide sequence of SEQ ID NO: 3 and any other nucleotide sequence to correspond as much as possible, analyzing the sequence, and The base sequence of SEQ ID NO: 3 and at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% This may mean having more than one sequence homology.
  • MyoG gene may refer to a gene encoding a muscle-specific transcription factor that can induce muscle production in various cell types of tissues. Myogenin can be expressed from the MyoG gene and can act as a transcriptional activator with roles in muscle differentiation, cell cycle, and muscle atrophy.
  • MyoD gene may refer to a gene encoding a muscle-specific transcription factor that plays an important role in regulating muscle differentiation.
  • the MyoD protein (or myoblast determination protein 1) can be expressed from the MyoD gene.
  • Myf6 gene may refer to a gene encoding a transcription factor of the myogenic factor (MRF) family known to be involved in the myogenic process.
  • MRF myogenic factor
  • Myf6 protein or Mrf4 or herculin can be expressed from the Myf6 gene.
  • Another example of the present invention relates to stem cells for muscle tissue regeneration.
  • stem cells for muscle tissue regeneration may be transformed with a vector containing a first nucleotide containing the MyoG gene, a second nucleotide containing the MyoD gene, and a third nucleotide containing the Myf6 gene.
  • stem cell refers to an undifferentiated cell that has the ability to self-replicate and differentiate into two or more different types of cells.
  • Stem cells may be autologous or allogeneic.
  • the stem cells may be one or more types of adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells, e.g.
  • it may be human tissue-derived mesenchymal stem cells, but is not limited thereto.
  • the mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta, for example, bone marrow. , but is not limited to this.
  • Another example of the present invention relates to a method for producing stem cells for muscle tissue regeneration comprising the following steps:
  • transfection refers to any mechanism that can incorporate a vector into a host cell. Successful transfection results in the host cell being capable of expressing all operable genes carried by the vector. Transfection can be stable or transient.
  • transient transfection involves expression of a vector within a specific cell, where the vector does not integrate within the host cell genome.
  • stable transfection may involve expression of the vector within a specific cell, where the vector is integrated into the host cell genome.
  • the transfection step is one or more methods selected from the group consisting of calcium phosphorylation transfection, microinjection, liposome injection, electroporation, gene gun, and jet injection. It may be performed, for example, by electroporation.
  • the electroporation method has the advantage of being able to optimize gene introduction efficiency by varying conditions depending on the state of the cell and the size and type of the gene.
  • Another example of the present invention relates to a pharmaceutical composition for preventing, improving or treating fibrotic diseases containing stem cells for muscle tissue regeneration.
  • fibrosis disease may mean loss of tissue function due to fibrosis, in which normal tissue is destroyed and replaced by fibrous connective tissue.
  • fibrotic diseases include esophagus, lungs, kidneys, liver, heart, blood vessels, joints, intestines, skin, soft tissues, bone marrow, penis, peritoneum, muscles, spine, testes, ovaries, breasts, thyroid gland, and eardrums. , pancreas, gallbladder, bladder, and prostate fibrosis. For example, it may be esophageal fibrosis, but is not limited thereto.
  • fibrotic disease may be caused by radiation irradiation.
  • stem cells for muscle tissue regeneration can promote the regeneration of muscle tissue by promoting the production and differentiation of muscle cells, and in particular, improve and prevent dysphagia by promoting the regeneration of esophageal muscle tissue. Or it can be treated.
  • the term “swallowing disorder” refers to a side effect that commonly occurs after radiation treatment in patients with head and neck cancer, where the tissue of the mucous membrane and muscle layer of the esophagus becomes fibrosed and the function of the esophagus deteriorates, making it difficult to swallow food or water.
  • the pharmaceutical composition of the present invention is a vector containing a first nucleotide containing the MyoG gene, a second nucleotide containing the MyoD gene, and a third nucleotide containing the Myf6 gene, and is a drug for transformed stem cells for muscle tissue regeneration. It can be used as a pharmaceutical composition containing a scientifically effective amount and/or a pharmaceutically acceptable carrier.
  • the term “pharmaceutically effective amount” refers to an amount sufficient to achieve the efficacy or activity of the stem cells for muscle tissue regeneration described above.
  • the cell therapy agent for application to esophageal tissue is 3 cells/mL , 3 cells/mL , 3 cells/mL , 3 cells/mL , 4 cells/mL , 4 cells/mL , 4 cells/mL , 4 cells / mL, 5 cells/mL , 5 cells/mL , 5 cells/mL , 5 It may be included at a concentration of cells/mL or 5
  • the pharmaceutical composition for application to esophageal tissue contains 3 4 to 7 x 10 7 cells/mL, 3 4 to 7 x 10 6 cells/mL, 3 4 to 7 x 10 5 cells/mL, 3 4 to 6 x 10 5 cells/mL, 4 4 to 7 x 10 7 cells/mL, 4 4 to 7 x 10 6 cells/mL, 4 4 to 7 x 10 5 cells/mL, 4 4 to 6 x 10 5 cells/mL, 5 4 to 7 x 10 7 cells/mL, 5 4 to 7 x 10 6 cells/mL, 5 4 to 7 x 10 5 cells/mL, 5 4 to 6 5 cells/mL or 5 5 5 It may be contained at a concentration of cells/mL, for example, 5 5 It may be included in cells/mL concentration, but is not limited to this.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work.
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.
  • the pharmaceutical composition according to the present invention can be administered to mammals, including humans, through various routes.
  • the administration method may be any commonly used method, for example, oral, dermal, intravenous, intramuscular, subcutaneous, etc.
  • the appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, body weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (e.g., hydrogel), and may additionally contain a dispersant or stabilizer. .
  • Another example of the present invention is a stem transfected with a vector containing at least one selected from the group consisting of a first nucleotide containing the MyoG gene, a second nucleotide containing the MyoD gene, and a third nucleotide containing the Myf gene. It may be a cell therapy product containing cells for the prevention, improvement, and treatment of fibrotic diseases.
  • cell therapy refers to the use of live autologous, allogenic, or xenogenic cells to proliferate or select in vitro or to determine the biological characteristics of cells by other methods in order to restore the function of cells and tissues. It may refer to a medicine used for the purpose of treatment, diagnosis, and prevention of fibrotic disease through a series of actions such as changing it.
  • the cell therapy agent can be administered through any general route as long as it can reach the target tissue, orally or parenterally, for example, intraperitoneal, intramuscularly ( It may be administered to a subject by one or more routes selected from the group including intramuscular, intravenous, subcutaneous, and intradermal, but is not limited thereto.
  • the cell therapy agent may be formulated in a suitable form with a pharmaceutical carrier commonly used in cell therapy.
  • a pharmaceutical carrier commonly used in cell therapy.
  • Pharmaceutically acceptable carriers include, for example, water, suitable oils, saline solutions, carriers for parenteral administration such as aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the cell therapy product may include a therapeutically effective amount of stem cells for the treatment of disease.
  • a 'therapeutically effective amount' refers to an active ingredient or pharmaceutical that induces a biological or medical response in a tissue system, animal, or human as considered by a researcher, veterinarian, physician, or other clinician.
  • the cell therapy agent included in the composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell therapy content can be easily determined by a person skilled in the art, depending on the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, gender, and diet. , can be adjusted according to various factors, including administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs. It is important to consider all of the above factors and include an amount that can achieve the maximum effect with the minimum amount without side effects.
  • the cell therapy agent may further include appropriate carriers, excipients, and diluents commonly used in the production of cell therapy compositions.
  • the cell therapy agent is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods.
  • Carriers, excipients, and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, and calcium.
  • a cell therapy product or composition when formulating a cell therapy product or composition, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations are made by mixing the above compound with at least one excipient, such as cotton, starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.
  • Liquid preparations for oral administration of the pharmaceutical composition include suspensions, oral solutions, emulsions, syrups, etc.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
  • As a base for suppositories wethepsol, macrogol, Tween 61, cacao, laurin, glycerol gelatin, etc. can be used.
  • Another example of the present invention is a vector containing a first nucleotide containing the MyoG gene, a second nucleotide containing the MyoD gene, and a third nucleotide containing the Myf6 gene, for use in muscle tissue regeneration of transformed stem cells. It's about.
  • the present invention relates to stem cells transfected with a vector co-introduced with MyoG, MyoD, and Myf6, which are genes for improving muscle differentiation, and a method for producing the same.
  • damaged muscle tissue can be regenerated, and in particular, two Radiation irradiation for cervical cancer treatment can improve the degree of damage to the damaged esophageal muscle layer, or prevent or treat damage.
  • Figure 1 is a schematic diagram showing a vector for producing stem cells for muscle tissue regeneration, a process for producing stem cells transfected with the same, and a process for applying the stem cells to esophageal tissue according to an experimental example of the present invention.
  • Figures 2a to 2e are fluorescence photographs confirming GFP expression in mesenchymal stem cells into which MyoD, MyoG, and Myf6 genes were individually or jointly transfected according to an experimental example of the present invention.
  • Figure 3 is a photograph of the overall appearance of esophageal tissue through H&E staining according to an experimental example of the present invention.
  • Figure 4 is a photograph taken by observing histological changes in the esophageal muscle layer for each group through desmin immunostaining according to an experimental example of the present invention.
  • Figure 5 is a graph showing fluorescence images and expression levels for confirming a-SMA expression according to an experimental example of the present invention.
  • Figure 6 is a photograph taken after Desmin immunostaining to confirm the thickness of the muscle layer according to an experimental example of the present invention.
  • Figure 7 is a photograph of the results of CD68 immunostaining according to an experimental example of the present invention.
  • Figure 8 is a graph showing the results of qPCR analysis of specific muscle gene expression patterns according to an experimental example of the present invention.
  • a vector for producing stem cells for muscle tissue regeneration comprising one or more nucleotides selected from the group consisting of a first nucleotide containing the MyoG gene, a second nucleotide containing the MyoD gene, and a third nucleotide containing the Myf6 gene.
  • pEGFPN1 vector a non-viral vector
  • the multi cloning site of the pEGFP N1 vector was cleaved with Xho1 and BamH1, respectively.
  • PCR was performed by adding Xho1 and BamH1 base sequences to both ends, respectively, and the amplified MyoG PCR product was cloned into the prepared vector.
  • MyoD gene was also amplified by PCR, PCR was performed by adding Xho1 and BamH1 base sequences to both ends, respectively, and the amplified MyoD PCR product was cloned into the prepared vector.
  • the vector pEGFPN1 was digested with HindIII and BamH1, respectively.
  • PCR was performed by adding Xho1 and BamH1 base sequences to both ends, respectively, and the amplified Myf6 PCR product was cloned into the prepared vector.
  • pEGFP N1-MyoD-MyoG-Myf6 was cloned by linking the three genes to the vector.
  • Figures 2A to 2E The results of transfecting MyoD, MyoG, and Myf6 genes separately or jointly into mesenchymal stem cells through electroporation and confirming GFP expression in the mesenchymal stem cells using a fluorescence microscope are shown in Figures 2A to 2E.
  • Figure 2a is a control group without the gene transfected
  • Figure 2b is a stem cell transfected with the MyoD gene
  • Figure 2c is a stem cell transfected with the MyoG gene
  • Figure 2d is a stem cell transfected with the Myf6 gene
  • Figure 2e is MyoD+MyoG+ This shows the fluorescence expression of stem cells transfected with all Myf6 genes.
  • IL-1 beta was significantly decreased in the three experimental groups (MyoD, Myf6, combination) compared to the control group.
  • MyoD MyoD
  • Myf6 Myf6, combination
  • TGF-beta known as a gene specific for tissue fibrosis
  • saline group a significant decrease compared to the saline group in all four experimental groups. This is presumed to be due to the anti-fibrosis effect of mesenchymal stem cells.
  • the distribution of macrophages expressed in the submucosal layer was quantitatively evaluated using CD-68 immunostaining and is shown in Figure 7 and Table 6.
  • the Myf5 gene a transcription factor
  • the Myf5 gene showed a similar tendency to the tissue staining results confirmed in Figure 4. Additionally, the expression of the Myogenin gene was significantly increased in the MyoG-group compared to the other two gene groups.
  • Calponin and SM22a representative genes related to smooth muscle, also showed significant differences in the group in which the three genes were co-introduced (MyoD+MyoG+Myf6) compared to other groups in which the genes were introduced alone. This supports the finding that the transfection of a gene that improves muscle differentiation ability affected the repair of muscle genes within the damaged DNA of the esophageal muscle layer and tissue regeneration.
  • the purpose of the present invention is to provide a vector for producing stem cells for muscle tissue regeneration containing MyoG, MyoD, and Myf6 genes.
  • Another object of the present invention is to provide stem cells for muscle tissue regeneration transfected with a vector carrying MyoG, MyoD and Myf6 genes.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing, improving or treating dysphagia.
  • Another object of the present invention is to provide a method for producing a composition for muscle tissue regeneration.
  • Another object of the present invention is to provide a use of stem cells containing MyoG, MyoD and Myf6 genes for muscle tissue regeneration.
  • SEQ ID NO: 1 is the sequence of the first nucleotide containing the MyoG gene.
  • SEQ ID NO: 2 is the sequence of the second nucleotide containing the MyoD gene.
  • SEQ ID NO: 3 is the sequence of the third nucleotide containing the Myf6 gene.

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Abstract

La présente invention concerne des cellules souches transfectées avec un vecteur dans lequel MyoG, MyoD et Myf6, qui sont des gènes pour améliorer la différenciation musculaire, sont co-introduits, et un procédé de préparation associé. Lors de l'utilisation de la présente invention, il est possible de régénérer un tissu musculaire endommagé, et en particulier, d'améliorer le degré d'endommagement d'une couche musculaire oesophagienne endommagée par une exposition à un rayonnement pour le traitement du cancer de la tête et du cou, ou de prévenir ou d'améliorer les dommages.
PCT/KR2023/018990 2022-11-25 2023-11-23 Composition d'agent thérapeutique cellulaire hautement fonctionnel pour régénérer des muscles endommagés par une radiothérapie WO2024112118A1 (fr)

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KR1020220160287A KR20240090030A (ko) 2022-11-25 2022-11-25 방사선 치료에 의하여 손상된 근육 재생을 위한 고기능성 세포 치료제 조성물
KR10-2022-0160287 2022-11-25

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150015076A (ko) * 2013-07-31 2015-02-10 한국생명공학연구원 ＭｙｏＤ 단백질을 포함하는 근육 분화 유도용 조성물
KR101496610B1 (ko) * 2014-03-04 2015-02-25 서울대학교산학협력단 줄기세포의 근육 세포로의 분화 유도용 조성물
KR20160092201A (ko) * 2015-01-27 2016-08-04 서울대학교산학협력단 근손상 질환의 치료용 조성물
KR20160115908A (ko) * 2013-10-25 2016-10-06 웨인 스테이트 유니버시티 단백질-유도 생체내 세포 재프로그래밍을 통한 세포 전환 방법, 시스템 및 조성물
KR20210102870A (ko) * 2018-08-30 2021-08-20 테나야 테라퓨틱스, 인코포레이티드 미오카르딘 및 ascl1을 사용한 심장 세포 재프로그래밍

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20150015076A (ko) * 2013-07-31 2015-02-10 한국생명공학연구원 ＭｙｏＤ 단백질을 포함하는 근육 분화 유도용 조성물
KR20160115908A (ko) * 2013-10-25 2016-10-06 웨인 스테이트 유니버시티 단백질-유도 생체내 세포 재프로그래밍을 통한 세포 전환 방법, 시스템 및 조성물
KR101496610B1 (ko) * 2014-03-04 2015-02-25 서울대학교산학협력단 줄기세포의 근육 세포로의 분화 유도용 조성물
KR20160092201A (ko) * 2015-01-27 2016-08-04 서울대학교산학협력단 근손상 질환의 치료용 조성물
KR20210102870A (ko) * 2018-08-30 2021-08-20 테나야 테라퓨틱스, 인코포레이티드 미오카르딘 및 ascl1을 사용한 심장 세포 재프로그래밍

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