WO2023284380A1 - Clinical application of cells as vectors for gene therapy - Google Patents

Clinical application of cells as vectors for gene therapy Download PDF

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WO2023284380A1
WO2023284380A1 PCT/CN2022/091281 CN2022091281W WO2023284380A1 WO 2023284380 A1 WO2023284380 A1 WO 2023284380A1 CN 2022091281 W CN2022091281 W CN 2022091281W WO 2023284380 A1 WO2023284380 A1 WO 2023284380A1
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cell therapy
cells
therapy according
rna
therapeutic
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PCT/CN2022/091281
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French (fr)
Chinese (zh)
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顾雨春
吴理达
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呈诺再生医学科技(北京)有限公司
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Priority claimed from CN202110801214.9A external-priority patent/CN113528526A/en
Priority claimed from CN202110919023.2A external-priority patent/CN113527519B/en
Application filed by 呈诺再生医学科技(北京)有限公司 filed Critical 呈诺再生医学科技(北京)有限公司
Publication of WO2023284380A1 publication Critical patent/WO2023284380A1/en

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    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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

Definitions

  • the invention belongs to the field of cell therapy and relates to the clinical application of cells as gene therapy carriers.
  • Gene therapy refers to the introduction of exogenous normal genes into appropriate recipient cells of patients through gene transfer technology, so that the products produced by exogenous genes can treat certain diseases to correct or compensate for diseases caused by defects and abnormal genes , in order to achieve the purpose of treatment.
  • it can be divided into gene correction and gene replacement, that is, to correct the abnormal sequence of the defective gene, and to repair the defective gene accurately in situ without any other changes in the genome.
  • the other type is gene augmentation and gene inactivation, which do not remove abnormal genes, but introduce foreign genes to make them express normal products, thereby compensating for the functions of defective genes, etc.; or specifically blocking certain The translation or transcription of genes to achieve the suppression of certain abnormal gene expression.
  • the introduced gene can be mRNA, miRNA, ncRNA, shRNA, gRNA, etc.
  • exosomes are used as the primary carrier to load mRNA, miRNA, ncRNA, shRNA, gRNA, etc. required for treatment.
  • Use autologous or allogeneic adult cells with high clinical safety or adult cell precursor cells or stem cells with low immune origin, such as umbilical cord-derived, adipose-derived, bone marrow-derived mesenchymal stem cells (MSC), dental pulp stem cells (TpSC), myelin sheath Precursor cells (OPCs) etc. are used as secondary carriers to produce specific exosomes.
  • the above cells are locally injected into the disease site or administered systemically through blood and lymphatic channels such as veins. Then adult cells or adult precursor cells are enriched around the disease through the mechanism of homing to the disease, and continuously produce and release exosomes. Exosomes enter target cells through specific targeting to achieve the introduction of therapeutic genes and achieve the purpose of treatment. Because the cells have a certain survival period in the body, they are finally cleared by the body's immune system to achieve safety. During the survival period, the cells can continuously produce exosomes, reaching an effective concentration in the local area of action without increasing the overall blood drug concentration of the body, while ensuring that the action lasts for a certain period of time.
  • the present invention provides a cell therapy comprising administering therapeutic cells capable of secreting extracellular vesicles or a cell therapy agent comprising the same to a person in need.
  • the therapeutic cells that secrete extracellular vesicles are derived from plants, microorganisms, animals, and humans.
  • therapeutic cells that secrete extracellular vesicles are derived from humans.
  • the therapeutic cells secreting extracellular vesicles include mature somatic cells or precursor cells of mature somatic cells.
  • the precursor cells of the mature somatic cells include stem cells.
  • the stem cells include totipotent stem cells, pluripotent stem cells, and unipotent stem cells.
  • cells of origin for therapeutically useful cells capable of secreting extracellular vesicles mentioned in the present invention include, but are not limited to, primary cells, cell lines, cells present in multicellular organisms, or substantially any other type of cell source .
  • Cells of the invention include cells that produce extracellular vesicles in vivo.
  • Cells according to the invention can be selected from a wide range of cells and cell lines, such as mesenchymal stem cells, dental pulp stem cells or stromal cells (which can be obtained from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue , placenta, dental buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amnion cells and more specifically amnion epithelial cells optionally expressing various early markers, myeloid suppressor cells, M2 polarized macrophages cells, adipocytes, endothelial cells, fibroblasts, etc.
  • mesenchymal stem cells such as mesenchymal stem cells, dental pulp stem cells or stromal cells (which can be obtained from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue , placenta, dental buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amn
  • Cell lines of particular interest include human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular endothelial cells or lymphatic endothelial cells, erythrocytes, erythroid progenitors, chondrocytes, MSCs of various origins, amnion cells, amnion epithelial (AE) cells, any cells obtained by amniocentesis or from placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, etc.
  • HEVEC human umbilical cord endothelial cells
  • HEK human embryonic kidney
  • endothelial cell lines such as microvascular endothelial cells or lymphatic endothelial cells
  • erythrocytes erythroid progenitors
  • chondrocytes chondrocytes
  • MSCs of various origins amnion cells
  • extracellular vesicles can be derived from essentially any cellular source, whether primary or immortalized cell lines.
  • Extracellular vesicle-derived cells can be any embryonic, fetal, and adult somatic stem cell type, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method.
  • iPSCs induced pluripotent stem cells
  • the cells may be allogeneic, autologous, or even xenogeneic in nature to the patient to be treated, ie the cells may be from the patient himself or from an unrelated, matched or mismatched donor.
  • extracellular vesicles include exosomes, vesicles, microvesicles, microparticles, endosome-derived vesicles, multivesicular bodies, apoptotic bodies, and combinations thereof.
  • extracellular vesicles are exosomes. The same applies to the following.
  • extracellular vesicles contain a therapeutic substance or a test substance for treating a disease suffered by a person in need thereof.
  • the extracellular vesicle also includes a recognition motif for an RNA-binding polypeptide or an RNA-binding polypeptide that directs the therapeutic substance or detection substance into the extracellular vesicle.
  • the recognition motif of the RNA-binding polypeptide is linked to the therapeutic substance or detection substance.
  • the recognition motif of the RNA-binding polypeptide is directly or indirectly linked to the therapeutic substance or detection substance.
  • RNA-binding polypeptide recognizes RNA through the recognition motif of the RNA-binding polypeptide.
  • RNA-binding polypeptide includes MS2 protein, hnRNPA2B1 protein or extracellular vesicle polypeptide.
  • RNA-binding polypeptide includes extracellular vesicle polypeptide and MS2 protein, and the extracellular vesicle polypeptide is linked to MS2 protein.
  • the C-terminal or N-terminal of the extracellular vesicle polypeptide is connected with MS2 protein.
  • the C-terminus of the extracellular vesicle polypeptide is connected with MS2 protein.
  • extracellular vesicle polypeptides include extracellular vesicle membrane proteins or fragments thereof, proteins in extracellular vesicles or fragments thereof.
  • the extracellular vesicle polypeptide includes the following proteins or fragments thereof: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, AARDC1, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptors, interleukin receptors, immunoglobulins, MHC- I or MHC-II components, CD2, CD3 ⁇ , CD3 ⁇ , CD13,
  • the recognition motif of the RNA-binding polypeptide includes the following sequence: G-X-Y-G, wherein X is G, A, U, and Y is A, U.
  • the recognition motif of the RNA-binding polypeptide includes the following sequence: M-G-X-Y-G, wherein, M is A, U; X is G, A, U, and Y is A, U.
  • the recognition motif of the RNA-binding polypeptide includes the following sequence: N-M-G-X-Y-G, wherein, M is A, U; N is A, C, G, U; X is G, A, U, and Y is A, U.
  • the recognition motif of the RNA-binding polypeptide includes the following sequence: N-M-G-X-Y-G, wherein, M is A, C, G, U; N is A, C, G, U; X is G, A, U, and Y is A, U.
  • the recognition motif of the RNA-binding polypeptide includes the following sequence: L-N-M-G-X-Y-G, wherein, M is A, C, G, U; N is A, C, G, U; L is A, C, G, U; X is G, A, U, Y is A, U.
  • the recognition motif of the RNA-binding polypeptide includes the sequence shown in SEQ ID NO.8.
  • the above-mentioned therapeutic substance of the present invention changes the phenotype and physiological state of diseased cells in the body of a person in need.
  • the aforementioned therapeutic substances of the present invention include polynucleotides, proteins, polypeptides, and small molecules.
  • polynucleotides include coding RNA, non-coding RNA, and DNA.
  • non-coding RNA includes ncRNA, shRNA, siRNA, miRNA, gRNA.
  • polynucleotides, proteins, polypeptides, and small molecules of the present invention are contained or not contained in naturally occurring extracellular vesicles.
  • polynucleotides, proteins, polypeptides, and small molecules of the present invention are not contained in naturally occurring extracellular vesicles.
  • Therapeutic proteins that can be used in the present invention include the following: antibodies, intrabodies, single chain variable fragments (scFv), affibodies, bispecific and multispecific antibodies or conjugates, receptors, ligands , enzymes for e.g.
  • enzyme replacement therapy or gene editing tumor suppressors, viral or bacterial inhibitors, cellular component proteins, DNA and/or RNA binding proteins, DNA repair inhibitors, nucleases, proteases, integrases, transcription Factors, growth factors, apoptosis inhibitors and inducers, toxins (such as Pseudomonas exotoxin), structural proteins, neurotrophic factors (such as NT3/4), brain-derived neurotrophic factor (BDNF), and nerve growth factors (NGF) and its individual subunits (such as 2.5S ⁇ subunit), ion channels, membrane transporters, protein homeostasis factors, proteins involved in cell signaling, translation and transcription-related proteins, nucleotide-binding proteins, protein-binding proteins , lipid-binding proteins, glycosaminoglycans (GAG) and GAG-binding proteins, metabolic proteins, cellular stress regulatory proteins, inflammation and immune system regulatory proteins, mitochondrial proteins, and heat shock proteins, etc.
  • toxins such as Pseudom
  • the detection substances include fluorescent labels, colorimetric labels, photochromic compounds, magnetic particles or other chemical labels.
  • the detection substance can be biotin or His tag.
  • the fluorescent label can be a fluorophore.
  • the extracellular vesicle of the present invention also includes a targeting polypeptide, and the targeting polypeptide recognizes a target cell membrane protein.
  • the targeting polypeptide includes proteins, peptides, single-chain antibodies or any other derivatives of antibodies.
  • targeting polypeptide is linked to extracellular vesicle membrane protein or its transmembrane domain or membrane-associated domain.
  • the sequential manner may be direct connection, or various linkers, release domains or release sites, cleavage sites or cleavage domains may be used to connect and/or attach to each other.
  • a cleavage linker is introduced such that it is possible to cleave (eg, by enzymatic activity) the cleavage linker and thereby separate the two ligated parts.
  • Suitable examples of cleavage linkers are TEV linkers or SUMO linkers, which can be cleaved by two different types of proteases, respectively.
  • the release domain enables the release of the therapeutic substance once it has been loaded into extracellular vesicles such as exosomes.
  • a suitable example of such a release linker is an intron.
  • Suitable extracellular vesicle membrane proteins or their transmembrane domains or membrane-associated domains may be selected from the group comprising: CD63, CD81, CD9, CD82, CD44, CD47, CD55, LAMP2B, ICAM, integrins, ARRDC1, Annexin, and any other extracellular vesicle polypeptide, and any combination, derivative, domain or region thereof.
  • one of the methods for obtaining therapeutic cells that secrete extracellular vesicles includes the following steps:
  • step 2) contacting the extracellular vesicles obtained in step 1) with cells;
  • step 2) Cultivate and purify the cells in step 2) to obtain cells that secrete extracellular vesicles containing therapeutic substances or test substances, and obtain therapeutic cells that secrete extracellular vesicles.
  • Polynucleotides can be introduced directly into exosomes or other vesicular structures described herein by using any suitable technique, examples of which include, but are not limited to, electroporation, incubation, cell activation and transfection, lipotransfer transfection, lipid delivery, liposome delivery, polymer transfection, polymer delivery, delivery by peptides (i.e. but not limited to cationic peptides, amphiphilic peptides, cell penetrating peptides), calcium or magnesium precipitation and ionic precipitation ( Also known as DNA-calcium phosphate precipitation).
  • peptides i.e. but not limited to cationic peptides, amphiphilic peptides, cell penetrating peptides
  • calcium or magnesium precipitation and ionic precipitation also known as DNA-calcium phosphate precipitation.
  • one of the two methods for obtaining therapeutic cells that secrete extracellular vesicles includes the following steps:
  • the step of introducing it into cells that secrete extracellular vesicles includes;
  • RNA polynucleotide comprising the aforementioned RNA-binding polypeptide recognition motif into a cell
  • Step i) and step ii) are in no particular order.
  • the step of introducing it into cells that secrete extracellular vesicles includes;
  • Step I) and step I) are in no particular order.
  • Extracellular vesicle-producing cells can be genetically modified with at least one polynucleotide construct using essentially any non-viral or viral method for introducing polynucleotides into cells.
  • a polynucleotide can be introduced into an extracellular vesicle-producing cell using essentially any non-viral or viral method for introducing a polynucleotide into a cell.
  • Suitable methods for introducing polynucleotides include transfection using polycations such as PEI, lipid-based transfection reagents such as liposomes (RTM), lentiviral transduction, CRISPR-Cas guided insertion , Flp-In system, transposon system, electroporation, DEAE-dextran transfection and calcium phosphate transfection.
  • polycations such as PEI
  • lipid-based transfection reagents such as liposomes (RTM)
  • lentiviral transduction such as liposomes (RTM)
  • CRISPR-Cas guided insertion CRISPR-Cas guided insertion
  • Flp-In system Flp-In system
  • transposon system transposon system
  • electroporation DEAE-dextran transfection and calcium phosphate transfection.
  • DEAE-dextran transfection calcium phosphate transfection.
  • linear DNA polynucleotide or mRNA linear DNA polynucleotide or mRNA
  • desired level of compliance and control production of extracellular vesicles can be achieved using techniques well known in the field of cell line development, including hTERT-mediated immortalization, transcription factor immortalization, E1/E2 immortalization, or other virus-mediated immortalization techniques Immortalization of cells to generate stable cell lines.
  • the cell therapeutic agent of the present invention comprises the therapeutic cells and a pharmaceutically acceptable carrier.
  • the therapeutic cells that secrete extracellular vesicles can be formulated in a suitable form together with a carrier that is a pharmaceutically acceptable carrier commonly used in cell therapy. Therefore, the above-mentioned cell therapy agent of the present invention comprises therapeutic cells secreting extracellular vesicles and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and generally does not cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans.
  • pharmaceutically acceptable carriers include water, suitable oils, physiological saline, water-soluble glucose, ethylene glycol, and other parenteral administration carriers, and may further include stabilizers and preservatives.
  • Suitable stabilizers are antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives are benzalkonium chloride, methyl or propyl paraben, chlorobutanol.
  • the cell therapy agent of the present invention is usually used in the form of parenteral preparations such as injections.
  • carriers that can be used in parenteral preparations include aqueous carriers such as physiological saline, isotonic solutions containing glucose, D-sorbitol, and the like.
  • the injections of the present invention include injections, such as physiological saline, lactated Ringer's solution, compound electrolyte injection, 5% glucose injection, 20% HSA injection, succinylated gelatin injection, succinylated gelatin MIX injection, MZJ Injection 1, MZJ Injection 2, MZJ Injection 3, Human Serum Protein Injection, Bomaili A, Potassium Chloride Injection, Magnesium Sulfate Injection, Sodium Bicarbonate Injection, Glucose Sodium Chloride Injection, Compound sodium chloride injection (Ringer's solution), dextran 20 glucose injection (small molecule), amino acid injection, hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch 40 sodium chloride injection, hydroxy Ethyl Starch 40 Sodium Chloride Injection, Low Molecular Weight Heparin Calcium for Injection, Heparin Sodium Injection, Coenzyme A for Injection, Cytidine Triphosphate Disodium, Lysine Hydro
  • the injection contains an isotonic or hypertonic solution; preferably, the solution is selected from NaCl injection (such as 0.9% to 2.7% NaCl injection), glucose injection (such as 4% to 5% glucose injection), Sodium lactate Ringer injection, compound electrolyte injection, HSA injection (eg 10%-20% HSA injection), succinylated gelatin injection (eg 4%-5% succinylated gelatin injection) and any combination thereof.
  • NaCl injection such as 0.9% to 2.7% NaCl injection
  • glucose injection such as 4% to 5% glucose injection
  • Sodium lactate Ringer injection compound electrolyte injection
  • HSA injection eg 10%-20% HSA injection
  • succinylated gelatin injection eg 4%-5% succinylated gelatin injection
  • the cell therapy agent of the present invention can also contain one or more injection additives in addition to the injection, for example selected from solubilizers, wetting agents, emulsifiers, buffers, suspending agents, chelating agents, antioxidants, pH Antimicrobial agents, local anesthetics, isotonicity regulators, fillers, protective agents and any combination thereof.
  • injection additives for example selected from solubilizers, wetting agents, emulsifiers, buffers, suspending agents, chelating agents, antioxidants, pH Antimicrobial agents, local anesthetics, isotonicity regulators, fillers, protective agents and any combination thereof.
  • the cell therapeutic agent is a mixture of the therapeutic cells and physiological saline.
  • the cell therapy agent is an intravenous injection.
  • the dosage of the cell therapy agent is positively correlated with the body weight of the subject.
  • the administration method of the therapeutic agent is:
  • the therapeutic cells of each therapeutic dose are dissolved in 100 ml of physiological saline; 0.1-0.2 ml of the cell therapy agent is intravenously injected at a rate of 60 drops per minute.
  • the cell therapy agent also includes any one or more of the following functional components:
  • the functional components include serum substitutes, non-essential amino acids, glutamine, stabilized dipeptides of L-alanyl-L-glutamine, growth factors and any combination thereof.
  • the cell therapy agent of the present invention can be administered via any conventional route so long as it reaches the target tissue.
  • cellular therapeutics can be administered by any device capable of delivering the active ingredient to target cells.
  • the cell therapy agent of the present invention is a parenteral preparation, for example, local administration such as intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, subcutaneous administration, subcapsular administration, etc. Medicine (capsule means membrane tissue covering various organs), intrathecal administration.
  • the cell therapy agent of the present invention is administered to a living body by intravenous administration.
  • the cellular therapeutic agents of the present invention may be administered in a therapeutically effective amount, as used herein, the phrase "therapeutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to any medical treatment. Effective dosage levels may vary depending on a variety of factors including the type, severity, age and sex of the individual, drug activity, drug sensitivity, time of administration, route of administration, discharge ratio, Treatment period and co-administered drugs and other factors well known in the medical art.
  • the dose of the cell therapy agent is not less than 1 ⁇ 10 4 cells/mL (for example, not less than 1 ⁇ 10 4 cells/ml, not less than 3 ⁇ 10 4 cells/ml, Not less than 5 ⁇ 10 4 /ml, not less than 7 ⁇ 10 4 /ml, not less than 1 ⁇ 10 5 /ml, not less than 3 ⁇ 10 5 /ml, not less than 5 ⁇ 10 5 pieces/ml, not less than 7 ⁇ 10 5 pieces/ml, not less than 1 ⁇ 10 6 pieces/ml, not less than 3 ⁇ 10 6 pieces/ml, not less than 5 ⁇ 10 6 pieces/ml, no Less than 7 ⁇ 10 6 /ml, not less than 1 ⁇ 10 6 /ml, not less than 3 ⁇ 10 6 /ml, not less than 5 ⁇ 10 6 /ml, not less than 7 ⁇ 10 6 pcs/ml, not less than 1 ⁇ 10 6 pcs/ml, not less than 3 ⁇ 10 6 pcs/ml, not
  • the dose of the cell therapy agent is not less than 1 ⁇ 10 3 cells/kg (for example, not less than 1 ⁇ 10 3 cells/kg, not less than 3 ⁇ 10 3 cells/kg, Not less than 5 ⁇ 10 3 pieces/kg, not less than 7 ⁇ 10 3 pieces/kg, not less than 1 ⁇ 10 4 pieces/kg, not less than 3 ⁇ 10 4 pieces/kg, not less than 5 ⁇ 10 4 pieces/kg, not less than 7 ⁇ 10 4 pieces/kg, not less than 1 ⁇ 10 5 pieces/kg, not less than 3 ⁇ 10 5 pieces/kg, not less than 5 ⁇ 10 5 pieces/kg, no Less than 7 ⁇ 10 5 pieces/kg, not less than 1 ⁇ 10 6 pieces/kg, not less than 3 ⁇ 10 6 pieces/kg, not less than 5 ⁇ 10 6 pieces/kg, not less than 7 ⁇ 10 6 pieces/kg pieces/kg, not less than 1 ⁇ 107 pieces/kg, not less than 3 ⁇ 107 pieces/kg, not less than 5 ⁇ 107 pieces/kg, not less than 7 ⁇ 107 pieces/kg, not less Less
  • the cellular therapeutics of the invention can be administered alone or in combination with other therapies. Co-administration of a therapeutic agent of the invention with other therapies can be performed simultaneously or sequentially. Single or multiple doses are possible. It is important to use the smallest possible amount sufficient to obtain maximum benefit without side effects, all factors considered.
  • the diseases applicable to the cell therapy of the present invention include but not limited to: hereditary diseases, tumors, autoimmune diseases, nervous system diseases, cardiovascular diseases or gastrointestinal diseases.
  • Non-limiting examples of diseases of the invention include: Crohn's disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, sarcoidosis, idiopathic pulmonary fibrosis psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), endometriosis, autoimmune hepatitis, scleroderma, myositis , stroke, acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acute myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, meningitis, encephalitis, liver failure, nonalcoholic steatohepatitis (NASH) , nonalcoholic fatty liver disease (NAFLD), renal failure, heart failure, or any acute or chronic organ failure and related underlying etiologie
  • ALL acute lymphoblastic leukemia
  • acute myeloid leukemia acute myeloid leukemia
  • adrenocortical carcinoma AIDS-related cancer
  • AIDS-related lymphoma anal cancer
  • appendix cancer astrocytoma Cytoma, cerebellar or brain cancer
  • basal cell carcinoma cholangiocarcinoma, bladder cancer, bone tumor, brainstem glioma, brain cancer, brain tumor (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, Ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway hypothalamic glioma), breast cancer, bronchial adenoma/carcinoid, Burkitt lymphoma, carcinoid tumor (children, Gastrointestinal tract), unknown primary tumor, central nervous system lymphoma, cerebellar astrocytoma/gli
  • the "person in need” mentioned herein is the subject to be treated, which can be any animal or human being.
  • the subject is preferably a mammal, more preferably a human.
  • the subject may be a non-human mammal, but is more preferably a human.
  • the subject can be male or female.
  • the subject can be a patient.
  • Therapeutic use can be in humans or animals (veterinary use).
  • Target cells as used herein depend on the disease to be treated.
  • Fig. 1 shows the result graph of the infection efficiency of EGFP lentivirus once infected MSC cells
  • Figure 2 shows the results of the infection efficiency of EGFP lentivirus once infected dental pulp stem cells
  • Figure 3 shows the results of infection efficiency of MSC cells infected with EGFP lentivirus for three times
  • Fig. 4 shows the infection efficiency result diagram of EGFP lentivirus infection of dental pulp stem cells for three times
  • Figure 5 shows the result graph of infection efficiency after MSC passage three times
  • Figure 6 shows the result graph of the infection efficiency of dental pulp stem cells after passage three times
  • Figure 7 shows the statistical diagram of the ratio of EGFP mRNA in dental pulp stem cells and MSC cell exosomes to cytoplasm
  • Figure 8 shows the statistical results of the expression levels of EGFP mRNA in various tissues and organs of animals after injection of dental pulp stem cells
  • Figure 9 shows the statistical results of the expression levels of EGFP mRNA in various tissues and organs of animals after MSC injection
  • Figure 10 shows a schematic diagram of the structure of the pLenti-EF1 ⁇ -Lamp2b-MS2 vector
  • Figure 11 shows a schematic diagram of the structure of the pLenti-EF1 ⁇ -MS2-Neo vector
  • Figure 12 shows a schematic diagram of the structure of the pLenti-EF1 ⁇ -RVG-Lamp2b-MS2 vector
  • Figure 13 shows the fluorescence image after exosomes infect cells
  • Figure 14 shows a schematic diagram of the structure of the pLKO.1-miR-151a-5p-puro lentiviral vector
  • Figure 15 shows a schematic diagram of the structure of the pLKO.1-miR-155-5p-puro lentiviral vector
  • Figure 16 shows a schematic diagram of the structure of the pLV-EF1a-HNRNPA2B1-IRES-Hygro lentiviral vector
  • Figure 17 shows the expression level results of miR-151a-5p and miR-155-5p in exosomes
  • Figure 18 shows the results of expression levels of miR-155-5p and its mutants in exosomes.
  • the present disclosure includes all polynucleotide and amino acid sequences described herein. Every RNA sequence includes its DNA equivalent, and every DNA sequence includes its RNA equivalent. Complementary and antiparallel polynucleotide sequences are included. This disclosure encompasses every DNA and RNA sequence that encodes a polypeptide disclosed herein. The present disclosure includes polynucleotide consensus sequences and motifs.
  • the backbone vector pCDH-MSCV-MCS-EF1-Puro was purchased from SBI (article number: CD710B-1);
  • the lentiviral vector constructed in this example was named pCDH-MSCV-EGFP-T2A-CD63-MS2-EF1-Puro.
  • the gene sequence is as follows:
  • the medium used for MSC and dental pulp stem cells is the same, and the medium formula is shown in Table 1.
  • 1Resuscitation Take out the frozen MSCs and dental pulp stem cells from the liquid nitrogen tank, place them in a 37°C water bath to thaw quickly, add 1ml MEM ⁇ medium to the cryopreservation tube to resuspend, centrifuge at 1300r/min for 5min, and remove the supernatant , resuspended with MEM ⁇ medium and plated in a 10cm cell culture dish, cultured at 37°C, 5% CO 2 .
  • RNA Isolation Mini Kit Use the Cell Culture Media Exosome Purification and RNA Isolation Mini Kit (norgenbiotek, Cat. No.: Cat. 60700) to extract the exosomes in the above cell supernatant, and extract the total RNA, and take an appropriate amount of total RNA for reverse transcription.
  • the reverse transcription kit is II Reverse Transcriptase (full gold, AH101-02), reverse transcription was performed according to the manufacturer's instructions.
  • PCR SuperMix whole gold, AS111-11 was used for PCR, and the reaction system described in Table 3 was premixed.
  • Reverse primer 5'-CTTGTAGTTGCCGTCGTCCTTGAA-3' (SEQ ID NO.7).
  • the experimental group was administered at the rate of 2 million cells/kg body weight (resuspended cells were prepared with physiological saline), and the experimental group was reinfused with the same volume of normal saline.
  • the main organs (heart, liver, spleen, lung, kidney, brain, testis, ovary, small intestine, stomach) were collected, total RNA was extracted, and the expression of EGFP mRNA in the organs was detected by qPCR.
  • Fig. 9 After the injection of mesenchymal stem cells, the expression levels of EGFP mRNA in various tissues and organs of the animals at different time points are shown in Fig. 9 .
  • autologous or allogeneic mature somatic cells or precursor cells of mature somatic cells are infected by lentivirus to load target therapeutic genes in exosomes, including mRNA, miRNA, ncRNA, shRNA, gRNA, etc.
  • the cells are cultured for one generation and then infused intravenously into the body.
  • the test found that the introduced gene can be detected in different tissues and organs in the animal body.
  • the amount of EGFP-containing mRNA produced by dental pulp stem cells and MSCs at different time points was measured, which proved that using cells as carriers can continuously and effectively target cells. Delivery of the introduced gene.
  • This embodiment involves two vectors, one vector (as shown in Figure 10) expresses proteins including Lamp2b (or PDGFT), and its N-terminus (inserted by the BsmBI restriction site) can express proteins including but not limited to recognizing neuronal cells RVG protein, or GE11 protein that recognizes EGFR receptors, or other specific single-chain antibodies, etc., its C-terminus expresses MS2 protein, which is used to recognize mRNA containing acaugaggaucacccaug (SEQ ID NO.8) sequence; another vector expresses mRNA containing For the mRNA of acaugaggaucacccaug sequence, take GFP as an example, as shown in Figure 11, GFP is inserted by BsiWI and NheI restriction sites, and can be replaced with other target genes or RNA sequences.
  • Lamp2b or PDGFT
  • N-terminus inserted by the BsmBI restriction site
  • MS2 protein which is used to recognize mRNA containing acaugaggaucaccc
  • the exosome membrane protein vector pLenti-EF1 ⁇ -Lamp2b-MS2 (nucleotide sequence shown in SEQ ID NO.9) is expressed.
  • This vector can be used to package lentivirus and contains puromycin selection marker, vector Contains EF1 ⁇ promoter to promote the expression of Lamp2b-MS2 fusion protein.
  • the N-terminal membrane signal of Lamp2b protein is designed with a BsmBI restriction site, which can be seamlessly connected to, for example, the RVG protein that recognizes neuron cells.
  • the vector contains WPRE components, Can increase the corresponding protein expression.
  • the target mRNA carrier containing the MS2 recognition sequence (acaugaggaucacccaug) (taking EGFP as an example, pLenti-EF1 ⁇ -EGFP-MS2-Neo carrier, the nucleotide sequence is shown in SEQ ID NO.10), which can be The mRNA sequence of other target genes can be connected into other target gene mRNA sequences for protein expression through BsiWI and NheI restriction sites.
  • miRNA200cluster (miRNA200, miRNA200b, miRNA200c, miRNA141, miRNA429)
  • the vector contains NeoR resistance gene
  • G418 can be used for resistance screening
  • the vector can also be used to package lentivirus.
  • RVG protein vector containing the recognition of neuron cells as shown in Figure 12, pLenti-EF1 ⁇ -RVG-Lamp2b-MS2, the nucleotide sequence of RVG is shown in SEQ ID NO.11
  • infect HEK293T Cells were screened with puromycin to obtain a stably transfected cell line (referred to as 293T-RVG-Lamp2b-MS2).
  • step 2) Pick the monoclonal in step 2) and expand the culture of 293T-RVG-Lamp2b-MS2-EGFP cells, collect the supernatant, concentrate the exosomes, use the concentrated exosomes to infect the cells, and observe the fluorescence.
  • the pLKO.1-puro plasmid (sigma, Cat. No. SHC001) is used as the backbone vector for miRNA expression, and the restriction sites are AgeI and EcoRI.
  • hsa-miR-151a-5p is UCGAGGAGCUCACAGUCUAGU (SEQ ID NO: 12), GGAG sequence exists in hsa-miR-151a-5;
  • hsa-miR-155-5p The sequence of hsa-miR-155-5p is UUAAUGCUAAUCGUGAUAGGGGUU (SEQ ID NO: 13), and there is no GGAG sequence in hsa-miR-155-5p.
  • shRNA structural sequence of hsa-miR-151a-5p is:
  • shRNA structural sequence of hsa-miR-155-5p is:
  • HNRNPA2B1 Gene synthesis of the HNRNPA2B1 sequence (its nucleotide sequence is shown in SEQ ID NO: 16, and its amino acid sequence is shown in SEQ ID NO: 17), the synthesis company is Anhui General Biotechnology Co., Ltd., and the HNRNPA2B1 sequence is inserted into pLV-EF1a - Between EcoRI and Hpa1 of the IRES-Hygro plasmid, the vector is named pLV-EF1a-HNRNPA2B1-IRES-Hygro (the schematic diagram of the vector is shown in Figure 16).
  • Cell inoculation inoculate 1.5 ⁇ 10 7 293T cells in a 10cm dish. Add 10ml of DMEM (Thermo Fisher Scientific, 11965084) medium containing 10% FBS (hyclone, SH30084.03), culture overnight at 37°C in a 5% CO 2 incubator, and transfect after 16-24 hours;
  • DMEM Thermo Fisher Scientific, 11965084
  • FBS hyclone, SH30084.03
  • Transfection medium After 16-18 hours, remove the medium containing the transfection reagent, add 10ml of DMEM containing 10% FBS, 5% CO 2 , and continue to culture at 37°C (at this time, the cell supernatant will produce virus).
  • the second virus harvest Harvest the cell supernatant, transfer it to a 50ml centrifuge tube, centrifuge at 3,000rpm for 10min, filter the supernatant with a 0.45 ⁇ m filter membrane, and store at 4°C. Cells were discarded after being treated with 10% disinfectant (84 disinfectant).
  • Virus concentration filter the collected lentivirus components with a 0.45 ⁇ m filter to remove bacterial contamination, mix the filtered components with Lenti-XTM Concentrator (clonetech, 631232) at a volume ratio of 3:1, and gently invert to mix .
  • exosome isolation reagent After centrifugation, remove the supernatant and resuspend with total exosome isolation reagent to obtain exosomes, which can be stored at 2-8 degrees for 1 week.
  • the shRNA structural sequence of mutant 1 is:
  • the shRNA structural sequence of mutant 2 is:
  • the shRNA structural sequence of mutant 3 is:
  • the shRNA structural sequence of mutant 4 is:
  • the shRNA structural sequence of mutant 5 is:
  • the shRNA structural sequence of mutant 6 is:
  • the shRNA structural sequence of mutant 7 is:
  • the shRNA structural sequence of mutant 8 is:

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Abstract

Provided is a clinical application of cells as vectors for gene therapy. Exosomes are used as primary vectors to load RNAs required for treatment, autologous or allogeneic mature somatic cells having high clinical safety or precursor cells of mature somatic cells having low immunogenicity are used as secondary vectors to load specific exosomes, and the cells are injected into a body for treatment. According to the cell therapy method, cells can be enriched around a disease, continuously generate and release exosomes, and the exosomes enter target cells by means of a specific targeting function, so as to achieve the purpose of introducing therapeutic genes to achieve treatment. The cell therapy method has good clinical application prospects due to its safety and effectiveness.

Description

细胞作为基因治疗的载体在临床中的应用Clinical Application of Cells as Carriers of Gene Therapy 技术领域technical field
本发明属于细胞治疗领域，涉及细胞作为基因治疗的载体在临床中的应用。The invention belongs to the field of cell therapy and relates to the clinical application of cells as gene therapy carriers.
背景技术Background technique
基因治疗(gene therapy)是指将外源正常基因通过基因转移技术导入病人的适当的受体细胞中，使外源基因制造的产物能治疗某种疾病以纠正或补偿缺陷和异常基因引起的疾病，以达到治疗目的。按照基因操作的类别可分为基因修正(gene correction)和基因置换(gene replacement)，即将缺陷基因的异常序列进行矫正，对缺陷基因精确地原位修复，不涉及基因组的其他任何改变。另一类为基因增强(gene augmentation)和基因失活(gene inactivation)，是不去除异常基因，而通过导入外源基因使其表达正常产物，从而补偿缺陷基因等的功能；或特异封闭某些基因的翻译或转录，以达到抑制某些异常基因表达。导入的基因可以为mRNA,miRNA,ncRNA,shRNA，gRNA等。Gene therapy refers to the introduction of exogenous normal genes into appropriate recipient cells of patients through gene transfer technology, so that the products produced by exogenous genes can treat certain diseases to correct or compensate for diseases caused by defects and abnormal genes , in order to achieve the purpose of treatment. According to the category of genetic manipulation, it can be divided into gene correction and gene replacement, that is, to correct the abnormal sequence of the defective gene, and to repair the defective gene accurately in situ without any other changes in the genome. The other type is gene augmentation and gene inactivation, which do not remove abnormal genes, but introduce foreign genes to make them express normal products, thereby compensating for the functions of defective genes, etc.; or specifically blocking certain The translation or transcription of genes to achieve the suppression of certain abnormal gene expression. The introduced gene can be mRNA, miRNA, ncRNA, shRNA, gRNA, etc.
目前基因导入细胞的常用方法如下：1.化学法；2.电穿孔法(electroportion)；3.显微注射法(microinjection)；4.脂质体法(liposome)；5.同源重组法(homologus recombination)；6.病毒介导基因转移。这些方法在体外把基因转导到细胞内过程可控方便，但上述方法应用到临床时面临重重困难。化学法，电穿孔，显微注射，同源重组法在病人层面都不容易操作，而脂质体法以及病毒介导的方法也因为安全性的问题，成本高昂，严重影响了基因治疗方法在临床中的尝试。到目前为止，基因携带的方法也只有AAV病毒和腺病毒被批准使用。如何使用更安全，临床上可控的方法，把要导入的基因包括mRNA,miRNA,ncRNA,shRNA，gRNA等有效并且大量的导入靶细胞中是亟待解决的问题。At present, the commonly used methods for gene introduction into cells are as follows: 1. chemical method; 2. electroporation method (electroportion); 3. microinjection method (microinjection); 4. liposome method (liposome); homologus recombination); 6. Virus-mediated gene transfer. These methods are controllable and convenient to transduce genes into cells in vitro, but face many difficulties when the above methods are applied clinically. Chemical methods, electroporation, microinjection, and homologous recombination methods are not easy to operate at the patient level, and liposome methods and virus-mediated methods are also costly due to safety issues, which seriously affect the use of gene therapy methods. clinical trials. So far, only AAV virus and adenovirus have been approved for gene delivery. How to use a safer and clinically controllable method to effectively and massively introduce the genes to be introduced into target cells, including mRNA, miRNA, ncRNA, shRNA, and gRNA, is an urgent problem to be solved.
发明内容Contents of the invention
为了解决现有技术存在的问题，本发明提供了一种新的细胞疗法：采用外泌体作为一级载体装载治疗所需的mRNA，miRNA，ncRNA，shRNA，gRNA等。采用临床安全性高的自体或异体成体细胞或免疫源低的成体细胞前体细胞或干细胞如脐带来源、脂肪来源、骨髓来源的间充质干细胞(MSC)，牙髓干细胞(TpSC)，髓鞘前体细胞(OPC)等作为二级载体产生特定的外泌体。在治疗过程中，把以上细胞局部注射到疾患部位或通过静脉等血液淋巴液途径进行全身给药。然后成体细胞或成体前体细胞通过对疾患归巢的作用机制富集在疾患周围，不停产生释放外泌体。外泌体通过特定的靶向作用进入靶细胞从而达到导入治疗基因，达到治疗的目的。由于细胞在体内有一定的存活期，最后被机体免疫***清除，实现安全性。在存活期内，细胞可以连续不断的产生外泌体，在作用局部达到了有效浓度，而不提高机体整体血药浓度，同时保证了作用在一定期间内持续。In order to solve the problems existing in the prior art, the present invention provides a new cell therapy: exosomes are used as the primary carrier to load mRNA, miRNA, ncRNA, shRNA, gRNA, etc. required for treatment. Use autologous or allogeneic adult cells with high clinical safety or adult cell precursor cells or stem cells with low immune origin, such as umbilical cord-derived, adipose-derived, bone marrow-derived mesenchymal stem cells (MSC), dental pulp stem cells (TpSC), myelin sheath Precursor cells (OPCs) etc. are used as secondary carriers to produce specific exosomes. During the course of treatment, the above cells are locally injected into the disease site or administered systemically through blood and lymphatic channels such as veins. Then adult cells or adult precursor cells are enriched around the disease through the mechanism of homing to the disease, and continuously produce and release exosomes. Exosomes enter target cells through specific targeting to achieve the introduction of therapeutic genes and achieve the purpose of treatment. Because the cells have a certain survival period in the body, they are finally cleared by the body's immune system to achieve safety. During the survival period, the cells can continuously produce exosomes, reaching an effective concentration in the local area of action without increasing the overall blood drug concentration of the body, while ensuring that the action lasts for a certain period of time.
根据本发明的一个方面，本发明提供了一种细胞疗法，所述细胞疗法包括给有需要者施用可以分泌细胞外囊泡的治疗用的细胞或包含其的细胞治疗剂。According to one aspect of the present invention, the present invention provides a cell therapy comprising administering therapeutic cells capable of secreting extracellular vesicles or a cell therapy agent comprising the same to a person in need.
进一步，所述分泌细胞外囊泡的治疗用的细胞来源于植物、微生物、动物、人类。Further, the therapeutic cells that secrete extracellular vesicles are derived from plants, microorganisms, animals, and humans.
进一步，所述分泌细胞外囊泡的治疗用的细胞来源于人类。Further, the therapeutic cells that secrete extracellular vesicles are derived from humans.
进一步，所述分泌细胞外囊泡的治疗用的细胞包括成熟体细胞或成熟体细胞的前体细胞。Further, the therapeutic cells secreting extracellular vesicles include mature somatic cells or precursor cells of mature somatic cells.
进一步，所述成熟体细胞的前体细胞包括干细胞。Further, the precursor cells of the mature somatic cells include stem cells.
进一步，所述干细胞包括全能干细胞、多能干细胞、单能干细胞。Further, the stem cells include totipotent stem cells, pluripotent stem cells, and unipotent stem cells.
具体地，本发明提到的能够分泌细胞外囊泡的治疗用的细胞的来源细胞包括但不限于原代细胞、细胞系、存在于多细胞生物的细胞、或基本上任何其它类型的细胞来源。本发明的细胞包括体内产生细胞外囊泡的细胞。根据本发明的细胞可以选自广泛范围的细胞和细胞系，例如间充质干细胞、牙髓干细胞或基质细胞(可从例如骨髓、脂肪组织、脐带胶质 (Wharton'sjelly)、围产期组织、胎盘、牙芽、脐带血、皮肤组织等获得)、成纤维细胞、羊膜细胞且更具体地说是任选地表达各种早期标记物的羊膜上皮细胞、骨髓抑制细胞、M2极化巨噬细胞、脂肪细胞、内皮细胞、成纤维细胞等。特别关注的细胞系包括人脐带内皮细胞(HUVEC)、人胚肾(HEK)细胞、内皮细胞系例如微血管内皮细胞或淋巴内皮细胞、红细胞、红系祖细胞、软骨细胞、不同来源的MSC、羊膜细胞、羊膜上皮(AE)细胞、通过羊膜穿刺术或从胎盘获得的任何细胞、气道上皮细胞或肺泡上皮细胞、成纤维细胞、内皮细胞等。此外，免疫细胞例如B细胞、T细胞、NK细胞、巨噬细胞、单核细胞、树突细胞(DC)也在本发明的范围内，并且基本上能够产生细胞外囊泡的任何类型的细胞也包括在本文中。通常，细胞外囊泡基本上可以源自任何细胞来源，无论是原始细胞来源还是永生化细胞系。细胞外囊泡源细胞可以是任何胚胎的、胎儿的和成年的体干细胞类型，包括诱导多能干细胞(iPSC)和通过任何方法衍生的其它干细胞。当治疗神经***疾病时，可以考虑利用例如原代神经细胞、星形胶质细胞、少突胶质细胞、小胶质细胞和神经祖细胞作为源细胞。对于待治疗的患者而言，细胞本质上可以是同种异体的、自体的、甚至异种的，即细胞可以来自患者本人或者来自无关的、匹配的或不匹配的供体。Specifically, cells of origin for therapeutically useful cells capable of secreting extracellular vesicles mentioned in the present invention include, but are not limited to, primary cells, cell lines, cells present in multicellular organisms, or substantially any other type of cell source . Cells of the invention include cells that produce extracellular vesicles in vivo. Cells according to the invention can be selected from a wide range of cells and cell lines, such as mesenchymal stem cells, dental pulp stem cells or stromal cells (which can be obtained from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue , placenta, dental buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amnion cells and more specifically amnion epithelial cells optionally expressing various early markers, myeloid suppressor cells, M2 polarized macrophages cells, adipocytes, endothelial cells, fibroblasts, etc. Cell lines of particular interest include human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular endothelial cells or lymphatic endothelial cells, erythrocytes, erythroid progenitors, chondrocytes, MSCs of various origins, amnion cells, amnion epithelial (AE) cells, any cells obtained by amniocentesis or from placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, etc. In addition, immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DCs) are also within the scope of the invention, and essentially any type of cell capable of producing extracellular vesicles Also included in this article. In general, extracellular vesicles can be derived from essentially any cellular source, whether primary or immortalized cell lines. Extracellular vesicle-derived cells can be any embryonic, fetal, and adult somatic stem cell type, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method. When treating diseases of the nervous system, utilization of, for example, primary neural cells, astrocytes, oligodendrocytes, microglia, and neural progenitor cells can be considered as source cells. The cells may be allogeneic, autologous, or even xenogeneic in nature to the patient to be treated, ie the cells may be from the patient himself or from an unrelated, matched or mismatched donor.
进一步，细胞外囊泡包括外泌体、囊泡、微囊泡、微颗粒、内体来源的囊泡、多囊泡体、凋亡小体及其组合。Further, extracellular vesicles include exosomes, vesicles, microvesicles, microparticles, endosome-derived vesicles, multivesicular bodies, apoptotic bodies, and combinations thereof.
进一步，细胞外囊泡是外泌体。以下同样如此限定。Further, extracellular vesicles are exosomes. The same applies to the following.
进一步，细胞外囊泡包含治疗有需要者所患疾病的治疗性物质或检测性物质。Further, the extracellular vesicles contain a therapeutic substance or a test substance for treating a disease suffered by a person in need thereof.
所述细胞外囊泡还包括RNA结合多肽的识别基序或RNA结合多肽，所述RNA结合多肽的识别基序引导所述治疗性物质或检测性物质进入细胞外囊泡。The extracellular vesicle also includes a recognition motif for an RNA-binding polypeptide or an RNA-binding polypeptide that directs the therapeutic substance or detection substance into the extracellular vesicle.
进一步，所述RNA结合多肽的识别基序与所述治疗性物质或检测性物质连接。Further, the recognition motif of the RNA-binding polypeptide is linked to the therapeutic substance or detection substance.
进一步，所述RNA结合多肽的识别基序与所述治疗性物质或检测性物质直接连接或间接连接。Further, the recognition motif of the RNA-binding polypeptide is directly or indirectly linked to the therapeutic substance or detection substance.
进一步，所述RNA结合多肽通过所述RNA结合多肽的识别基序识别RNA。Further, the RNA-binding polypeptide recognizes RNA through the recognition motif of the RNA-binding polypeptide.
进一步，所述RNA结合多肽包括MS2蛋白、hnRNPA2B1蛋白或细胞外囊泡多肽。Further, the RNA-binding polypeptide includes MS2 protein, hnRNPA2B1 protein or extracellular vesicle polypeptide.
进一步，所述RNA结合多肽包括细胞外囊泡多肽和MS2蛋白、细胞外囊泡多肽与MS2蛋白连接。Further, the RNA-binding polypeptide includes extracellular vesicle polypeptide and MS2 protein, and the extracellular vesicle polypeptide is linked to MS2 protein.
进一步，细胞外囊泡多肽的C端或N端与MS2蛋白连接。Further, the C-terminal or N-terminal of the extracellular vesicle polypeptide is connected with MS2 protein.
进一步，细胞外囊泡多肽的C端与MS2蛋白连接。Further, the C-terminus of the extracellular vesicle polypeptide is connected with MS2 protein.
进一步，细胞外囊泡多肽包括细胞外囊泡膜蛋白或其片段、细胞外囊泡内的蛋白或其片段。Further, extracellular vesicle polypeptides include extracellular vesicle membrane proteins or fragments thereof, proteins in extracellular vesicles or fragments thereof.
进一步，所述细胞外囊泡多肽包括以下蛋白或其片段：CD9、CD53、CD63、CD81、CD54、CD50、FLOT1、FLOT2、CD49d、CD71、CD133、CD138、CD235a、ALIX、AARDC1、Syntenin-1、Syntenin-2、Lamp2b、TSPAN8、syndecan-1、syndecan-2、syndecan-3、syndecan-4、TSPAN14、CD37、CD82、CD151、CD231、CD102、NOTCH1、NOTCH2、NOTCH3、NOTCH4、DLL1、DLL4、JAG1、JAG2、CD49d/ITGA4、ITGB5、ITGB6、ITGB7、CD11a、CD11b、CD11c、CD18/ITGB2、CD41、CD49b、CD49c、CD49e、CD51、CD61、CD104、Fc受体、白介素受体、免疫球蛋白、MHC-I或MHC-II成分、CD2、CD3ε、CD3ζ、CD13、CD18、CD19、CD30、CD34、CD36、CD40、CD40L、CD44、CD45、CD45RA、CD47、CD86、CD110、CD111、CD115、CD117、CD125、CD135、CD184、CD200、CD279、CD273、CD274、CD362、COL6A1、AGRN、EGFR、GAPDH、GLUR2、GLUR3、HLA-DM、HSPG2、L1CAM、LAMB1、LAMC1、LFA-1、LGALS3BP、Mac-1α、Mac-1β、MFGE8、SLIT2、STX3、TCRA、TCRB、TCRD、TCRG、VTI1A、VTI1B。Further, the extracellular vesicle polypeptide includes the following proteins or fragments thereof: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, AARDC1, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptors, interleukin receptors, immunoglobulins, MHC- I or MHC-II components, CD2, CD3ε, CD3ζ, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135 , CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1α, Mac-1β , MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B.
作为一些实施例，所述RNA结合多肽的识别基序包括以下序列：G-X-Y-G，其中X为G、A、U，Y为A、U。As some embodiments, the recognition motif of the RNA-binding polypeptide includes the following sequence: G-X-Y-G, wherein X is G, A, U, and Y is A, U.
更进一步，所述RNA结合多肽的识别基序包括以下序列：M-G-X-Y-G，其中，M为A、U；X为G、A、U，Y为A、U。Furthermore, the recognition motif of the RNA-binding polypeptide includes the following sequence: M-G-X-Y-G, wherein, M is A, U; X is G, A, U, and Y is A, U.
更进一步，所述RNA结合多肽的识别基序包括以下序列：N-M-G-X-Y-G，其中，M为A、U；N为A、C、G、U；X为G、A、U，Y为A、U。Furthermore, the recognition motif of the RNA-binding polypeptide includes the following sequence: N-M-G-X-Y-G, wherein, M is A, U; N is A, C, G, U; X is G, A, U, and Y is A, U.
更进一步，所述RNA结合多肽的识别基序包括以下序列：N-M-G-X-Y-G，其中，M为A、C、G、U；N为A、C、G、U；X为G、A、U，Y为A、U。Furthermore, the recognition motif of the RNA-binding polypeptide includes the following sequence: N-M-G-X-Y-G, wherein, M is A, C, G, U; N is A, C, G, U; X is G, A, U, and Y is A, U.
更进一步，所述RNA结合多肽的识别基序包括以下序列：L-N-M-G-X-Y-G，其中，M为A、C、G、U；N为A、C、G、U；L为A、C、G、U；X为G、A、U，Y为A、U。Further, the recognition motif of the RNA-binding polypeptide includes the following sequence: L-N-M-G-X-Y-G, wherein, M is A, C, G, U; N is A, C, G, U; L is A, C, G, U; X is G, A, U, Y is A, U.
作为另一些实施例，所述RNA结合多肽的识别基序包括SEQ ID NO.8所示的序列。As other embodiments, the recognition motif of the RNA-binding polypeptide includes the sequence shown in SEQ ID NO.8.
本发明前面所述的治疗性物质改变有需要者体内病变细胞的表型、生理状态。The above-mentioned therapeutic substance of the present invention changes the phenotype and physiological state of diseased cells in the body of a person in need.
本发明前面所述的治疗性物质包括多核苷酸、蛋白质、多肽、小分子。The aforementioned therapeutic substances of the present invention include polynucleotides, proteins, polypeptides, and small molecules.
进一步，多核苷酸包括编码RNA、非编码RNA、DNA。Further, polynucleotides include coding RNA, non-coding RNA, and DNA.
更进一步，非编码RNA包括ncRNA、shRNA、siRNA、miRNA、gRNA。Furthermore, non-coding RNA includes ncRNA, shRNA, siRNA, miRNA, gRNA.
本发明的所述多核苷酸、蛋白质、多肽、小分子是天然存在的细胞外囊泡中含有的或者不含有的。The polynucleotides, proteins, polypeptides, and small molecules of the present invention are contained or not contained in naturally occurring extracellular vesicles.
进一步，本发明的所述多核苷酸、蛋白质、多肽、小分子是天然存在的细胞外囊泡中不含有的。Furthermore, the polynucleotides, proteins, polypeptides, and small molecules of the present invention are not contained in naturally occurring extracellular vesicles.
可用于本发明的治疗性蛋白质包括以下：抗体、胞内抗体(intrabodies)、单链可变片段(scFv)、亲和体、双特异性和多特异性抗体或结合物、受体、配体、用于例如酶替代疗法或基因编辑的酶、肿瘤抑制剂、病毒或细菌抑制剂、细胞组分蛋白、DNA和/或RNA结合蛋白、DNA修复抑制剂、核酸酶、蛋白酶、整合酶、转录因子、生长因子、凋亡抑制剂和诱导剂、毒素(例如假单胞菌外毒素)、结构蛋白、神经营养因子(诸如NT3/4)、脑源性神经营养因子(BDNF)和神经生长因子(NGF)及其个体亚单位(诸如2.5Sβ亚单位)、离子通道、膜转运蛋白、蛋白稳态因子、参与细胞信号传导的蛋白、翻译和转录相关蛋白、核苷酸结合蛋白、蛋白结合蛋白、脂质结合蛋白、糖胺聚糖(GAG)和GAG结合蛋白、代谢蛋白、细胞应激调节蛋白、炎症和免疫***调节蛋白、线粒体蛋白和热休克蛋白等。Therapeutic proteins that can be used in the present invention include the following: antibodies, intrabodies, single chain variable fragments (scFv), affibodies, bispecific and multispecific antibodies or conjugates, receptors, ligands , enzymes for e.g. enzyme replacement therapy or gene editing, tumor suppressors, viral or bacterial inhibitors, cellular component proteins, DNA and/or RNA binding proteins, DNA repair inhibitors, nucleases, proteases, integrases, transcription Factors, growth factors, apoptosis inhibitors and inducers, toxins (such as Pseudomonas exotoxin), structural proteins, neurotrophic factors (such as NT3/4), brain-derived neurotrophic factor (BDNF), and nerve growth factors (NGF) and its individual subunits (such as 2.5Sβ subunit), ion channels, membrane transporters, protein homeostasis factors, proteins involved in cell signaling, translation and transcription-related proteins, nucleotide-binding proteins, protein-binding proteins , lipid-binding proteins, glycosaminoglycans (GAG) and GAG-binding proteins, metabolic proteins, cellular stress regulatory proteins, inflammation and immune system regulatory proteins, mitochondrial proteins, and heat shock proteins, etc.
进一步，所述检测性物质包括荧光标记、比色标记、光致变色化合物、磁性颗粒或其他化学标记。检测性物质可以是生物素或His标签。Further, the detection substances include fluorescent labels, colorimetric labels, photochromic compounds, magnetic particles or other chemical labels. The detection substance can be biotin or His tag.
进一步，荧光标记可以是荧光团。Further, the fluorescent label can be a fluorophore.
本发明的所述细胞外囊泡还包括靶向多肽，所述靶向多肽识别靶细胞膜蛋白。The extracellular vesicle of the present invention also includes a targeting polypeptide, and the targeting polypeptide recognizes a target cell membrane protein.
进一步，所述靶向多肽包括蛋白质、肽、单链抗体或抗体的任何其它衍生物。Further, the targeting polypeptide includes proteins, peptides, single-chain antibodies or any other derivatives of antibodies.
进一步，所述靶向多肽与细胞外囊泡膜蛋白或其跨膜结构域或膜相关结构域连接。Further, the targeting polypeptide is linked to extracellular vesicle membrane protein or its transmembrane domain or membrane-associated domain.
所述连续的方式可以是直接连接，或者可以使用各种接头、释放结构域或释放位点、切割位点或切割结构域彼此连接和/或附着。例如，在某些实施例中，引入切割接头，从而使得能够切割(例如通过酶促活性)切割接头并且由此分开连接的两部分。切割接头的合适的示例是TEV接头或SUMO接头，它们可以分别被两种不同类型的蛋白酶切割。释放结构域使得治疗性物质一旦已经被装载到细胞外囊泡(诸如外泌体)中就释放。这样的释放接头的合适的示例是内含子。The sequential manner may be direct connection, or various linkers, release domains or release sites, cleavage sites or cleavage domains may be used to connect and/or attach to each other. For example, in certain embodiments, a cleavage linker is introduced such that it is possible to cleave (eg, by enzymatic activity) the cleavage linker and thereby separate the two ligated parts. Suitable examples of cleavage linkers are TEV linkers or SUMO linkers, which can be cleaved by two different types of proteases, respectively. The release domain enables the release of the therapeutic substance once it has been loaded into extracellular vesicles such as exosomes. A suitable example of such a release linker is an intron.
合适的细胞外囊泡膜蛋白或其跨膜结构域或膜相关结构域可以选自包含以下的群组：CD63、CD81、CD9、CD82、CD44、CD47、CD55、LAMP2B、ICAM、整联蛋白、ARRDC1、膜联蛋白以及任何其他细胞外囊泡多肽，以及其任何组合、衍生物、结构域或区域。Suitable extracellular vesicle membrane proteins or their transmembrane domains or membrane-associated domains may be selected from the group comprising: CD63, CD81, CD9, CD82, CD44, CD47, CD55, LAMP2B, ICAM, integrins, ARRDC1, Annexin, and any other extracellular vesicle polypeptide, and any combination, derivative, domain or region thereof.
本发明的细胞疗法中，所述分泌细胞外囊泡的治疗用的细胞的获取方法之一包括如下步骤：In the cell therapy of the present invention, one of the methods for obtaining therapeutic cells that secrete extracellular vesicles includes the following steps:
1)将治疗性物质或检测性物质导入细胞外囊泡中；1) introducing therapeutic substances or detection substances into extracellular vesicles;
2)将步骤1)获得的细胞外囊泡与细胞接触；2) contacting the extracellular vesicles obtained in step 1) with cells;
3)培养纯化步骤2)的细胞获得包含分泌含有治疗性物质或检测性物质的细胞外囊泡的细胞，获得分泌细胞外囊泡的治疗用的细胞。3) Cultivate and purify the cells in step 2) to obtain cells that secrete extracellular vesicles containing therapeutic substances or test substances, and obtain therapeutic cells that secrete extracellular vesicles.
可以通过使用任何合适的技术将多核苷酸直接引入本文所述的外泌体或其它囊泡结构中，所述技术的实例包括但不限于电穿孔、孵育、细胞活化和转染、脂质转染、脂质递送、脂质体递送、聚合物转染、聚合物递送、通过肽递送(即但不限于阳离子肽、两亲性肽、细胞穿透肽)、钙或镁沉淀和离子沉淀(也称为DNA-磷酸钙沉淀)。Polynucleotides can be introduced directly into exosomes or other vesicular structures described herein by using any suitable technique, examples of which include, but are not limited to, electroporation, incubation, cell activation and transfection, lipotransfer transfection, lipid delivery, liposome delivery, polymer transfection, polymer delivery, delivery by peptides (i.e. but not limited to cationic peptides, amphiphilic peptides, cell penetrating peptides), calcium or magnesium precipitation and ionic precipitation ( Also known as DNA-calcium phosphate precipitation).
本发明的细胞疗法中，所述分泌细胞外囊泡的治疗用的细胞的获取方法之一之二包括如下步骤：In the cell therapy of the present invention, one of the two methods for obtaining therapeutic cells that secrete extracellular vesicles includes the following steps:
a)将治疗性物质或检测性物质导入分泌细胞外囊泡的细胞中；a) introducing a therapeutic or test substance into cells that secrete extracellular vesicles;
b)培养纯化步骤1)获得的细胞，获得分泌细胞外囊泡的治疗用的细胞。b) culturing the cells obtained in the purification step 1) to obtain therapeutic cells secreting extracellular vesicles.
进一步，当治疗性物质是RNA多核苷酸时，将其导入分泌细胞外囊泡的细胞的步骤包括；Further, when the therapeutic substance is RNA polynucleotide, the step of introducing it into cells that secrete extracellular vesicles includes;
i)将表达包含前面所述的RNA结合多肽识别基序的RNA多核苷酸的载体导入细胞；i) introducing a vector expressing an RNA polynucleotide comprising the aforementioned RNA-binding polypeptide recognition motif into a cell;
ii)将表达hnRNPA2B1或其功能类似物的载体导入细胞；ii) introducing a vector expressing hnRNPA2B1 or a functional analog thereof into the cell;
步骤i)和步骤ii)不分先后。Step i) and step ii) are in no particular order.
当治疗性物质是RNA多核苷酸时，将其导入分泌细胞外囊泡的细胞的步骤包括；When the therapeutic substance is an RNA polynucleotide, the step of introducing it into cells that secrete extracellular vesicles includes;
I)将表达包含SEQ ID NO.1所示的所述RNA结合多肽识别基序的RNA多核苷酸的载体导入细胞；1) introducing a vector expressing an RNA polynucleotide comprising the RNA-binding polypeptide recognition motif shown in SEQ ID NO.1 into a cell;
II)将表达前面所述的RNA结合多肽的载体导入细胞；II) introducing the vector expressing the aforementioned RNA-binding polypeptide into the cell;
步骤I)和步骤I)不分先后。Step I) and step I) are in no particular order.
可以使用用于将多核苷酸引入到细胞中的基本上任何非病毒或病毒方法，用至少一种多核苷酸构建体对产生细胞外囊泡的细胞进行基因修饰。可以使用用于将多核苷酸引入到细胞中的基本上任何非病毒或病毒方法将多核苷酸引入到产生细胞外囊泡的细胞中。用于引入多核苷酸的合适的方法包括使用聚阳离子(诸如PEI)的转染、基于脂质(诸如脂质体(RTM))的转染试剂、慢病毒转导、CRISPR-Cas引导的***、Flp-In***、转座子***、电穿孔、DEAE-葡聚糖转染和磷酸钙转染。用于将多核苷酸引入到产生细胞外囊泡的细胞中的方法的选择将取决于各种参数，包括细胞源的选择、多核苷酸载体的性质和特征(例如，如果载体是质粒或小环或例如线性DNA多核苷酸或mRNA)，以及所需的顺应性和控制的水平。类似地，使用细胞系开发领域中众所周知的技术(包括hTERT介导的永生化、转录因子永生化、E1/E2永生化或其他病毒介导的永生化技术等)，可以实现产生细胞外囊泡的细胞的永生化，以产生稳定的细胞系。Extracellular vesicle-producing cells can be genetically modified with at least one polynucleotide construct using essentially any non-viral or viral method for introducing polynucleotides into cells. A polynucleotide can be introduced into an extracellular vesicle-producing cell using essentially any non-viral or viral method for introducing a polynucleotide into a cell. Suitable methods for introducing polynucleotides include transfection using polycations such as PEI, lipid-based transfection reagents such as liposomes (RTM), lentiviral transduction, CRISPR-Cas guided insertion , Flp-In system, transposon system, electroporation, DEAE-dextran transfection and calcium phosphate transfection. The choice of method for introducing polynucleotides into extracellular vesicle-producing cells will depend on various parameters, including the choice of cell source, the nature and characteristics of the polynucleotide vector (e.g., if the vector is a plasmid or small circular or e.g. linear DNA polynucleotide or mRNA), and the desired level of compliance and control. Similarly, production of extracellular vesicles can be achieved using techniques well known in the field of cell line development, including hTERT-mediated immortalization, transcription factor immortalization, E1/E2 immortalization, or other virus-mediated immortalization techniques Immortalization of cells to generate stable cell lines.
本发明的所述细胞治疗剂包含所述治疗用的细胞和药学上允许的载体。The cell therapeutic agent of the present invention comprises the therapeutic cells and a pharmaceutically acceptable carrier.
分泌细胞外囊泡的治疗用的细胞可以与载体一起以适合的形态进行剂型化，该载体为常用于细胞治疗的药学上允许的载体。故本发明的前面所述的细胞治疗剂包含分泌细胞外囊泡的治疗用的细胞和药学上允许的载体。所谓“药学上允许的”是指生理学上允许的、且向人类给药时通常不会引起胃肠障碍、眩晕等过敏反应或与此类似反应的组合物。作为药学上允许的载体，例如有：水、合适的油、生理盐水、水溶性葡萄糖及乙二醇等非口服给药用载体等，还可进一步包括稳定剂及保存剂。合适的稳定剂为，亚硫酸氢钠、亚硫酸钠或抗坏血酸等抗氧化剂。合适的保存剂为，苯扎氯铵、对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、氯丁醇。作为其它药学上允许的载体，可参考以下文献中记载的内容(Remington's Pharmaceutical Sciences,19th ed.,Mack Publishing Company,Easton,PA,1995)。The therapeutic cells that secrete extracellular vesicles can be formulated in a suitable form together with a carrier that is a pharmaceutically acceptable carrier commonly used in cell therapy. Therefore, the above-mentioned cell therapy agent of the present invention comprises therapeutic cells secreting extracellular vesicles and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and generally does not cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans. Examples of pharmaceutically acceptable carriers include water, suitable oils, physiological saline, water-soluble glucose, ethylene glycol, and other parenteral administration carriers, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl or propyl paraben, chlorobutanol. As other pharmaceutically acceptable carriers, reference can be made to the contents described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
本发明的细胞治疗剂通常以注射剂等非口服制剂的形态使用。作为非口服制剂中能够使用的载体，可列举例如生理盐水、含有葡萄糖、D-山梨糖醇等的等渗液等水性载体。The cell therapy agent of the present invention is usually used in the form of parenteral preparations such as injections. Examples of carriers that can be used in parenteral preparations include aqueous carriers such as physiological saline, isotonic solutions containing glucose, D-sorbitol, and the like.
具体地，本发明注射剂中包含注射液，如生理盐水、乳酸钠林格液、复方电解质注射液、5％葡萄糖注射液、20％HSA注射液、琥珀酰明胶注射液、琥珀酰明胶MIX注射液、MZJ注射液1、MZJ注射液2、MZJ注射液3、人血清蛋白注射液、勃脉力A、氯化钾注射液、 硫酸镁注射液、碳酸氢钠注射液、葡萄糖氯化钠注射液、复方氯化钠注射液(林格氏液)、右旋糖酐20葡萄糖注射液(小分子)、氨基酸注射液、羟乙基淀粉40氯化钠注射液、羟乙基淀粉40氯化钠注射液、羟乙基淀粉40氯化钠注射液、注射用低分子量肝素钙、肝素钠注射液、注射用辅酶A、三磷酸胞苷二钠、注射用盐酸赖氨酸、维生素C注射液、胞磷胆碱氯化钠、注射用脂溶性维生素Ⅱ、注射用还原型谷胱甘肽、注射用脑蛋白水解物、脱氧核苷酸钠注射液、多种微量元素注射液Ⅱ、甘露醇注射液、盐酸精氨酸注射液、氯化钾注射液、注射用三磷酸胞苷二钠、注射用门冬氨酸鸟氨酸等。Specifically, the injections of the present invention include injections, such as physiological saline, lactated Ringer's solution, compound electrolyte injection, 5% glucose injection, 20% HSA injection, succinylated gelatin injection, succinylated gelatin MIX injection, MZJ Injection 1, MZJ Injection 2, MZJ Injection 3, Human Serum Protein Injection, Bomaili A, Potassium Chloride Injection, Magnesium Sulfate Injection, Sodium Bicarbonate Injection, Glucose Sodium Chloride Injection, Compound sodium chloride injection (Ringer's solution), dextran 20 glucose injection (small molecule), amino acid injection, hydroxyethyl starch 40 sodium chloride injection, hydroxyethyl starch 40 sodium chloride injection, hydroxy Ethyl Starch 40 Sodium Chloride Injection, Low Molecular Weight Heparin Calcium for Injection, Heparin Sodium Injection, Coenzyme A for Injection, Cytidine Triphosphate Disodium, Lysine Hydrochloride for Injection, Vitamin C Injection, Citicoline Sodium Chloride, Fat-soluble Vitamin II for Injection, Reduced Glutathione for Injection, Brain Protein Hydrolyzate for Injection, Sodium Deoxyribonucleotide Injection, Various Trace Elements Injection II, Mannitol Injection, Essence Hydrochloride Amino acid injection, potassium chloride injection, disodium cytidine triphosphate for injection, ornithine aspartate for injection, etc.
所述注射剂中含有等渗或高渗溶液；优选地，所述溶液选自NaCl注射液(例如0.9％～2.7％NaCl注射液)、葡萄糖注射液(例如4％～5％葡萄糖注射液)、乳酸钠林格注射液、复方电解质注射液、HSA注射液(例如10％～20％HSA注射液)、琥珀酰明胶注射液(例如4％～5％琥珀酰明胶注射液)及其任意组合。The injection contains an isotonic or hypertonic solution; preferably, the solution is selected from NaCl injection (such as 0.9% to 2.7% NaCl injection), glucose injection (such as 4% to 5% glucose injection), Sodium lactate Ringer injection, compound electrolyte injection, HSA injection (eg 10%-20% HSA injection), succinylated gelatin injection (eg 4%-5% succinylated gelatin injection) and any combination thereof.
本发明的细胞治疗剂除了注射液之外还可以含有一种或多种注射剂附加剂，例如选自增溶剂、湿润剂、乳化剂、缓冲剂、助悬剂、螯合剂、抗氧剂、抑菌剂、局麻剂、等渗调节剂、填充剂、保护剂及其任意组合。The cell therapy agent of the present invention can also contain one or more injection additives in addition to the injection, for example selected from solubilizers, wetting agents, emulsifiers, buffers, suspending agents, chelating agents, antioxidants, pH Antimicrobial agents, local anesthetics, isotonicity regulators, fillers, protective agents and any combination thereof.
在本发明的具体实施方案中，所述细胞治疗剂为所述治疗用的细胞与生理盐水的混合液。In a specific embodiment of the present invention, the cell therapeutic agent is a mixture of the therapeutic cells and physiological saline.
进一步，所述细胞治疗剂为静脉注射剂。Further, the cell therapy agent is an intravenous injection.
进一步，所述细胞治疗剂用量与用药对象的体重正相关。Further, the dosage of the cell therapy agent is positively correlated with the body weight of the subject.
进一步，所述治疗剂的用药方法是：Further, the administration method of the therapeutic agent is:
将每次治疗量的所述治疗用的细胞溶于100ml的生理盐水中；将0.1-0.2ml的细胞治疗剂以每分钟60滴的速度静脉注射完毕。The therapeutic cells of each therapeutic dose are dissolved in 100 ml of physiological saline; 0.1-0.2 ml of the cell therapy agent is intravenously injected at a rate of 60 drops per minute.
进一步，所述细胞治疗剂还包括下述功能性成分中的任意一种或多种：Further, the cell therapy agent also includes any one or more of the following functional components:
α)维持所述治疗用的细胞活性的成分；α) an ingredient that maintains the cellular activity of said therapy;
β)促进所述治疗用的细胞增殖的成分。β) An ingredient that promotes the proliferation of said therapeutic cells.
进一步，所述功能性成分包括血清替代物、非必需氨基酸、谷氨酰胺、L-丙氨酰-L-谷氨酰胺的稳定化二肽、生长因子及其任意组合。Further, the functional components include serum substitutes, non-essential amino acids, glutamine, stabilized dipeptides of L-alanyl-L-glutamine, growth factors and any combination thereof.
本发明的细胞治疗剂可以经由任何常规途径给药，只要它能够达到目标组织。另外，细胞治疗剂可以通过任何能能够将活性成分递送至靶细胞的设备给药。本发明的细胞治疗剂为非口服制剂的情况下，可以列举例如血管内给药(优选静脉内给药)、腹腔内给药、肠道内给药、皮下给药、被膜下给药等局部给药(被膜的意思是被覆各种器官的膜组织)、鞘内给药。一个优选实施方式中，本发明的细胞治疗剂通过静脉内给药而对生物体给药。The cell therapy agent of the present invention can be administered via any conventional route so long as it reaches the target tissue. In addition, cellular therapeutics can be administered by any device capable of delivering the active ingredient to target cells. When the cell therapy agent of the present invention is a parenteral preparation, for example, local administration such as intravascular administration (preferably intravenous administration), intraperitoneal administration, enteral administration, subcutaneous administration, subcapsular administration, etc. Medicine (capsule means membrane tissue covering various organs), intrathecal administration. In a preferred embodiment, the cell therapy agent of the present invention is administered to a living body by intravenous administration.
本发明的细胞治疗剂可以以治疗有效量给药，本文使用的短语“治疗有效量”是指足以以适用于任何医学治疗的合理利益/风险比治疗疾病的量。有效剂量水平可能因多种因素而异，所述多种因素包括个体的类型、严重程度、年纪和性别，药物活性、药物敏感度、给药时间、给药途径、排放系数(discharge ratio)、治疗周期和共给药的药物以及医学领域周知的其他因素。The cellular therapeutic agents of the present invention may be administered in a therapeutically effective amount, as used herein, the phrase "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to any medical treatment. Effective dosage levels may vary depending on a variety of factors including the type, severity, age and sex of the individual, drug activity, drug sensitivity, time of administration, route of administration, discharge ratio, Treatment period and co-administered drugs and other factors well known in the medical art.
在某些实施方案中，所述细胞治疗剂的给药剂量不少于1×10 4个/mL(例如不少于1×10 4个/ml，不少于3×10 4个/ml，不少于5×10 4个/ml，不少于7×10 4个/ml，不少于1×10 5个/ml，不少于3×10 5个/ml，不少于5×10 5个/ml，不少于7×10 5个/ml，不少于1×10 6个/ml，不少于3×10 6个/ml，不少于5×10 6个/ml，不少于7×10 6个/ml，不少于1×10 6个/ml，不少于3×10 6个/ml，不少于5×10 6个/ml，不少于7×10 6个/ml，不少于1×10 6个/ml，不少于3×10 6个/ml，不少于5×10 6个/ml，不少于7×10 6个/ml，不少于1×10 6个/ml，不少于3×10 6个/ml，不少于5×10 6个/ml，不少于7×10 6个/ml，不少于1×10 10个/ml，不少于3×10 10个/ml，不少于5×10 10个/ml或不少于7×10 10个/ml，又例如1×10 5-1×10 8、7×10 5-7×10 6、1×10 6-5×10 6个/ml。 In some embodiments, the dose of the cell therapy agent is not less than 1×10 4 cells/mL (for example, not less than 1×10 4 cells/ml, not less than 3×10 4 cells/ml, Not less than 5×10 4 /ml, not less than 7×10 4 /ml, not less than 1×10 5 /ml, not less than 3×10 5 /ml, not less than 5×10 5 pieces/ml, not less than 7×10 5 pieces/ml, not less than 1×10 6 pieces/ml, not less than 3×10 6 pieces/ml, not less than 5×10 6 pieces/ml, no Less than 7×10 6 /ml, not less than 1×10 6 /ml, not less than 3×10 6 /ml, not less than 5×10 6 /ml, not less than 7×10 6 pcs/ml, not less than 1×10 6 pcs/ml, not less than 3×10 6 pcs/ml, not less than 5×10 6 pcs/ml, not less than 7×10 6 pcs/ml, not less Less than 1×10 6 /ml, not less than 3×10 6 /ml, not less than 5×10 6 /ml, not less than 7×10 6 /ml, not less than 1×10 10 /ml, not less than 3×10 10 /ml, not less than 5×10 10 /ml or not less than 7×10 10 /ml, and for example 1×10 5 -1×10 8 , 7× 10 5 -7×10 6 , 1×10 6 -5×10 6 cells/ml.
在某些实施方案中，所述细胞治疗剂的给药剂量不少于1×10 3个/kg(例如不少于1×10 3个/kg，不少于3×10 3个/kg，不少于5×10 3个/kg，不少于7×10 3个/kg，不少于1×10 4个/kg，不少于3×10 4个/kg，不少于5×10 4个/kg，不少于7×10 4个/kg，不少于1×10 5个/kg，不 少于3×10 5个/kg，不少于5×10 5个/kg，不少于7×10 5个/kg，不少于1×10 6个/kg，不少于3×10 6个/kg，不少于5×10 6个/kg，不少于7×10 6个/kg，不少于1×10 7个/kg，不少于3×10 7个/kg，不少于5×10 7个/kg，不少于7×10 7个/kg，不少于1×10 8个/kg，不少于3×10 8个/kg，不少于5×10 8个/kg，不少于7×10 8个/kg，不少于1×10 9个/kg，不少于3×10 9个/kg，不少于5×10 9个/kg，不少于7×10 9个/kg，不少于1×10 10个/kg，不少于3×10 10个/kg，不少于5×10 10个/kg或不少于7×10 10个/kg，又例如1×10 5-1×10 8、7×10 5-7×10 6、1×10 6-5×10 6个/kg。 In some embodiments, the dose of the cell therapy agent is not less than 1×10 3 cells/kg (for example, not less than 1×10 3 cells/kg, not less than 3×10 3 cells/kg, Not less than 5×10 3 pieces/kg, not less than 7×10 3 pieces/kg, not less than 1×10 4 pieces/kg, not less than 3×10 4 pieces/kg, not less than 5×10 4 pieces/kg, not less than 7×10 4 pieces/kg, not less than 1×10 5 pieces/kg, not less than 3×10 5 pieces/kg, not less than 5×10 5 pieces/kg, no Less than 7×10 5 pieces/kg, not less than 1×10 6 pieces/kg, not less than 3×10 6 pieces/kg, not less than 5×10 6 pieces/kg, not less than 7×10 6 pieces/kg pieces/kg, not less than 1× 107 pieces/kg, not less than 3× 107 pieces/kg, not less than 5×107 pieces/kg, not less than 7 × 107 pieces/kg, not less Less than 1× 108 pieces/kg, not less than 3× 108 pieces/kg, not less than 5× 108 pieces/kg, not less than 7× 108 pieces/kg, not less than 1× 109 pieces /kg, not less than 3× 109 pieces/kg, not less than 5× 109 pieces/kg, not less than 7× 109 pieces/kg, not less than 1 ×1010 pieces/kg, not less than 3×10 10 pieces/kg, not less than 5×10 10 pieces/kg or not less than 7×10 10 pieces/kg, and for example 1×10 5 -1×10 8 , 7×10 5 -7×10 6. 1×10 6 -5×10 6 pieces/kg.
本发明的细胞治疗剂可以单独给予或与其他疗法联合给予。本发明的治疗剂与其他疗法的共给予可以同时地或者相继地进行。单个或者多个剂量均是可以的。重要的是，使用足以获得最大疗效而无副作用的最小可能量，在考虑所有因素的情况下。The cellular therapeutics of the invention can be administered alone or in combination with other therapies. Co-administration of a therapeutic agent of the invention with other therapies can be performed simultaneously or sequentially. Single or multiple doses are possible. It is important to use the smallest possible amount sufficient to obtain maximum benefit without side effects, all factors considered.
本发明的细胞疗法适用的疾病包括但不限于：遗传性疾病、肿瘤、自身免疫性疾病、神经***疾病、心血管疾病或胃肠道疾病。The diseases applicable to the cell therapy of the present invention include but not limited to: hereditary diseases, tumors, autoimmune diseases, nervous system diseases, cardiovascular diseases or gastrointestinal diseases.
本发明的疾病的非限制性实例包括：克罗恩病、溃疡性结肠炎、强直性脊柱炎、类风湿性关节炎、多发性硬化、***性红斑狼疮、结节病、特发性肺纤维化、牛皮癣、肿瘤坏死因子(TNF)受体相关周期性综合征(TRAPS)、白介素1受体拮抗剂缺乏症(DIRA)、子宫内膜异位症、自身免疫肝炎、硬皮病、肌炎、中风、急性脊髓损伤、血管炎、格林-巴利综合征、急性心肌梗塞、急性呼吸窘迫综合征(ARDS)、败血症、脑膜炎、脑炎、肝衰竭、非酒精性脂肪性肝炎(NASH)、非酒精性脂肪肝(NAFLD)、肾衰竭、心力衰竭或任何急性或慢性器官衰竭以及相关的潜在病因、移植物抗宿主病、Duchenne型肌营养不良症和其它肌肉萎缩症、溶酶体贮积症(例如戈谢病(Gaucher disease)、法布瑞氏症(Fabry's disease)、MPSI、II(亨特综合征(Hunter syndrome))和III、尼曼-匹克病(Niemann-Pick disease)A、B和C型、庞贝病(Pompe disease)等)、神经退行性疾病(包括阿尔茨海默病、帕金森病、亨廷顿病和其它三核苷酸重复相关疾病、痴呆、肌萎缩侧索硬化(ALS))、癌症引起的恶病质、厌食症、2型糖尿病和各种癌症。几乎所有类型的癌症都是本发明的相关疾病靶标，例如，急性淋巴细胞白血病(ALL)、急性髓细胞白血病、肾上腺皮质癌、艾滋病相关癌症、艾滋病相关淋巴瘤、***癌、阑尾癌、星形细胞瘤、小脑或脑癌、基底细胞癌、胆管癌、膀胱癌、骨肿瘤、脑干胶质瘤、脑癌、脑瘤(小脑星形细胞瘤、脑星形细胞瘤/恶性胶质瘤、室管膜瘤、成神经管细胞瘤、幕上原始神经外胚层瘤、视觉通路下丘脑胶质瘤)、乳腺癌、支气管腺瘤/类癌、伯基特淋巴瘤、类癌瘤(儿童、胃肠道)、原发灶不明肿瘤、中枢神经***淋巴瘤、小脑星形细胞瘤/恶性胶质瘤、***、慢性淋巴细胞白血病、慢性粒细胞白血病、慢性骨髓增殖性疾病、结肠癌、皮肤T细胞淋巴瘤、促***增生性小圆细胞瘤、子宫内膜癌、室管膜瘤、食道癌、颅外生殖细胞瘤、性腺外生殖细胞瘤、肝外胆管癌、眼癌(眼内黑色素瘤、视网膜母细胞瘤)、胆囊癌、胃癌、胃肠道类癌肿瘤、胃肠道间质瘤、生殖细胞瘤(颅外、性腺外或卵巢)、妊娠滋养细胞肿瘤、胶质瘤(脑干胶质瘤、脑星形细胞瘤、视觉通路下丘脑胶质瘤)、胃类癌、毛细胞白血病、头颈癌、心脏癌、肝细胞癌、霍奇金淋巴瘤、下咽癌、眼内黑色素瘤、胰岛细胞癌(内分泌胰腺)、卡波西肉瘤、肾癌(肾细胞癌)、喉癌、白血病(急性淋巴细胞白血病、急性髓细胞白血病、慢性淋巴细胞白血病、慢性髓细胞白血病、毛细胞白血病)、唇和口腔癌、脂肪肉瘤、肝癌(原发性)、肺癌(非小细胞、小细胞)、淋巴瘤、艾滋病相关淋巴瘤、布基特淋巴瘤、皮肤T细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤、成神经管细胞瘤、梅克尔细胞癌、间皮瘤、原发灶隐匿转移性鳞状颈癌、口腔癌、多发性内分泌肿瘤综合征、多发性骨髓瘤/浆细胞肿瘤、蕈样肉芽肿、骨髓增生异常/骨髓增生性疾病、骨髓性白血病、慢性粒细胞白血病(急性、慢性)、骨髓瘤、鼻腔和鼻窦癌、鼻咽癌、神经母细胞瘤、口腔癌、口咽癌、骨肉瘤/骨恶性纤维组织细胞瘤、卵巢癌、卵巢上皮癌(表面上皮间质瘤)、卵巢生殖细胞瘤、卵巢低恶性潜能瘤、胰腺癌、胰岛细胞癌、甲状旁腺癌、***癌、咽癌、嗜铬细胞瘤、松果体星形细胞瘤、松果体生殖细胞瘤、松果体母细胞瘤和幕上原始神经外胚层瘤、垂体腺瘤、胸膜肺母细胞瘤、***癌、直肠癌、肾细胞癌(肾癌)、视网膜母细胞瘤、横纹肌肉瘤、涎腺癌、肉瘤(尤因(Ewing)家族肿瘤肉瘤、卡波西肉瘤、软组织肉瘤、子宫肉瘤)、西泽里(Sezary)综合征、皮肤癌(非恶性肿瘤、黑色素瘤)、小肠癌、鳞状细胞癌、鳞状颈癌、胃癌、幕上原始神经外胚层肿瘤、睾丸癌、喉癌、胸腺瘤和胸腺癌、甲状腺癌、肾盂和输尿管移行细胞癌、尿道癌、子宫癌、子宫肉瘤、***癌、外阴癌、巨球蛋白血症和/或肾母细胞瘤。Non-limiting examples of diseases of the invention include: Crohn's disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, sarcoidosis, idiopathic pulmonary fibrosis psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), endometriosis, autoimmune hepatitis, scleroderma, myositis , stroke, acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acute myocardial infarction, acute respiratory distress syndrome (ARDS), sepsis, meningitis, encephalitis, liver failure, nonalcoholic steatohepatitis (NASH) , nonalcoholic fatty liver disease (NAFLD), renal failure, heart failure, or any acute or chronic organ failure and related underlying etiologies, graft-versus-host disease, Duchenne muscular dystrophy and other muscular dystrophies, lysosomal storage syndrome (such as Gaucher disease, Fabry's disease, MPSI, II (Hunter syndrome) and III, Niemann-Pick disease) A , types B and C, Pompe disease, etc.), neurodegenerative diseases (including Alzheimer's disease, Parkinson's disease, Huntington's disease and other trinucleotide repeat-related diseases, dementia, amyotrophic lateral sclerosis (ALS)), cancer-induced cachexia, anorexia, type 2 diabetes, and various cancers. Almost all types of cancer are relevant disease targets of the present invention, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma Cytoma, cerebellar or brain cancer, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumor, brainstem glioma, brain cancer, brain tumor (cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, Ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway hypothalamic glioma), breast cancer, bronchial adenoma/carcinoid, Burkitt lymphoma, carcinoid tumor (children, Gastrointestinal tract), unknown primary tumor, central nervous system lymphoma, cerebellar astrocytoma/glioblastoma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, colon cancer, Cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, eye cancer (eye Internal melanoma, retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor (extracranial, extragonadal, or ovarian), gestational trophoblastic tumor, glioma (brain stem glioma, brain astrocytoma, visual pathway hypothalamic glioma), gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, Intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), throat cancer, leukemia (acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia , hairy cell leukemia), lip and mouth cancer, liposarcoma, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma , Hodgkin's lymphoma, non-Hodgkin's lymphoma, medulloblastoma, Merkel cell carcinoma, mesothelioma, occult primary metastatic squamous neck carcinoma, oral cancer, multiple endocrine neoplasia syndrome , multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplasia/myeloproliferative disorder, myelogenous leukemia, chronic myelogenous leukemia (acute, chronic), myeloma, nasal cavity and sinus cancer, nasopharyngeal carcinoma, Neuroblastoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma (surface epithelial stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, Islet cell carcinoma, parathyroid carcinoma, penile carcinoma, pharyngeal carcinoma, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pinealoblastoma and supratentorial primitive neuroectodermal tumor, Pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma (Ewing family tumor sarcoma, Kaposi sarcomas, soft tissue sarcomas, uterine sarcomas), Sezary syndrome, skin cancers (non-malignant, melanoma), small bowel cancer, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymus carcinoma, thyroid cancer, renal pelvis and ureteral transitional cell carcinoma, urethra carcinoma, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, macroglobulinemia, and/or Wilms tumor.
文中所述“有需要者”是待治疗的受试者，可以是任何动物或人类。所述受试者优选是哺乳动物，更优选是人类。所述受试者可以是非人类哺乳动物，但更优选是人类。所述受试者可以是男性或女性。所述受试者可以是患者。治疗用途可用于人类或动物(兽医用途)。The "person in need" mentioned herein is the subject to be treated, which can be any animal or human being. The subject is preferably a mammal, more preferably a human. The subject may be a non-human mammal, but is more preferably a human. The subject can be male or female. The subject can be a patient. Therapeutic use can be in humans or animals (veterinary use).
文中所述“靶细胞”取决于待治疗的疾病。"Target cells" as used herein depend on the disease to be treated.
附图说明Description of drawings
图1显示EGFP慢病毒一次感染MSC细胞的感染效率结果图；Fig. 1 shows the result graph of the infection efficiency of EGFP lentivirus once infected MSC cells;
图2显示EGFP慢病毒一次感染牙髓干细胞的感染效率结果图；Figure 2 shows the results of the infection efficiency of EGFP lentivirus once infected dental pulp stem cells;
图3显示EGFP慢病毒三次感染MSC细胞的感染效率结果图；Figure 3 shows the results of infection efficiency of MSC cells infected with EGFP lentivirus for three times;
图4显示EGFP慢病毒三次感染牙髓干细胞的感染效率结果图；Fig. 4 shows the infection efficiency result diagram of EGFP lentivirus infection of dental pulp stem cells for three times;
图5显示MSC传代三次后的感染效率结果图；Figure 5 shows the result graph of infection efficiency after MSC passage three times;
图6显示牙髓干细胞传代三次后的感染效率结果图；Figure 6 shows the result graph of the infection efficiency of dental pulp stem cells after passage three times;
图7显示EGFP mRNA在牙髓干细胞和MSC细胞外泌体中与胞质中比例的统计图；Figure 7 shows the statistical diagram of the ratio of EGFP mRNA in dental pulp stem cells and MSC cell exosomes to cytoplasm;
图8显示注射牙髓干细胞后EGFP的mRNA在动物的各个组织器官表达水平的统计结果图；Figure 8 shows the statistical results of the expression levels of EGFP mRNA in various tissues and organs of animals after injection of dental pulp stem cells;
图9显示注射MSC后EGFP的mRNA在动物的各个组织器官表达水平的统计结果图；Figure 9 shows the statistical results of the expression levels of EGFP mRNA in various tissues and organs of animals after MSC injection;
图10显示pLenti-EF1α-Lamp2b-MS2载体的结构示意图；Figure 10 shows a schematic diagram of the structure of the pLenti-EF1α-Lamp2b-MS2 vector;
图11显示pLenti-EF1α-MS2-Neo载体的结构示意图；Figure 11 shows a schematic diagram of the structure of the pLenti-EF1α-MS2-Neo vector;
图12显示pLenti-EF1α-RVG-Lamp2b-MS2载体的结构示意图；Figure 12 shows a schematic diagram of the structure of the pLenti-EF1α-RVG-Lamp2b-MS2 vector;
图13显示外泌体侵染细胞后的荧光图；Figure 13 shows the fluorescence image after exosomes infect cells;
图14显示pLKO.1-miR-151a-5p-puro慢病毒载体结构示意图；Figure 14 shows a schematic diagram of the structure of the pLKO.1-miR-151a-5p-puro lentiviral vector;
图15显示pLKO.1-miR-155-5p-puro慢病毒载体结构示意图；Figure 15 shows a schematic diagram of the structure of the pLKO.1-miR-155-5p-puro lentiviral vector;
图16显示pLV-EF1a-HNRNPA2B1-IRES-Hygro慢病毒载体结构示意图；Figure 16 shows a schematic diagram of the structure of the pLV-EF1a-HNRNPA2B1-IRES-Hygro lentiviral vector;
图17显示miR-151a-5p和miR-155-5p在外泌体中的表达水平结果图；Figure 17 shows the expression level results of miR-151a-5p and miR-155-5p in exosomes;
图18显示miR-155-5p及其突变体在外泌体中的表达水平结果图。Figure 18 shows the results of expression levels of miR-155-5p and its mutants in exosomes.
具体实施方式detailed description
除非本文另外定义，否则本公开内容中使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的相同含义。Unless otherwise defined herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
在整个说明书中给出的每个数值范围包括其上限值和下限值，以及落入该范围内的每个较窄的数值范围，如同这些较窄的数值范围均在本文中明确写出。Every numerical range given throughout this specification includes its upper and lower numerical limits, and every narrower numerical range falling within that range, as if such narrower numerical ranges were all expressly written herein. .
本公开内容包括本文所述的所有多核苷酸和氨基酸序列。每个RNA序列包括其DNA等同物，并且每个DNA序列包括其RNA等同物。包括互补和反平行多核苷酸序列。本公开内容涵盖编码本文公开的多肽的每个DNA和RNA序列。本公开内容包括多核苷酸共有序列和基序。The present disclosure includes all polynucleotide and amino acid sequences described herein. Every RNA sequence includes its DNA equivalent, and every DNA sequence includes its RNA equivalent. Complementary and antiparallel polynucleotide sequences are included. This disclosure encompasses every DNA and RNA sequence that encodes a polypeptide disclosed herein. The present disclosure includes polynucleotide consensus sequences and motifs.
实施例1Example 1
I、EGFP表达质粒转染多能干细胞I. Transfection of EGFP expression plasmid into pluripotent stem cells
一、步骤1. Steps
1、EGFP的质粒构建1. Plasmid construction of EGFP
1)实验材料1) Experimental materials
骨架载体pCDH-MSCV-MCS-EF1-Puro购买自SBI(货号：CD710B-1)；The backbone vector pCDH-MSCV-MCS-EF1-Puro was purchased from SBI (article number: CD710B-1);
基因合成公司：安徽通用生物科技有限公司。Gene synthesis company: Anhui General Biotechnology Co., Ltd.
2)构建方法2) Construction method
在本实施例中构建得到的慢病毒载体命名为pCDH-MSCV-EGFP-T2A-CD63-MS2-EF1-Puro。The lentiviral vector constructed in this example was named pCDH-MSCV-EGFP-T2A-CD63-MS2-EF1-Puro.
通过基因合成，合成EGFP-T2A-CD63-MS2序列，基因序列如下：Through gene synthesis, synthesize the EGFP-T2A-CD63-MS2 sequence, the gene sequence is as follows:
EGFP序列：EGFP sequence:
Figure PCTCN2022091281-appb-000001
Figure PCTCN2022091281-appb-000001
T2A序列：T2A sequence:
Figure PCTCN2022091281-appb-000002
Figure PCTCN2022091281-appb-000002
CD63序列：CD63 sequence:
Figure PCTCN2022091281-appb-000003
Figure PCTCN2022091281-appb-000003
MS2序列：MS2 sequence:
Figure PCTCN2022091281-appb-000004
Figure PCTCN2022091281-appb-000004
在骨架载体pCDH-MSCV-MCS-EF1-Puro的NheⅠ(3`端)和NotⅠ(5`端)***基因合成的EGFP-T2A-CD63-MS2(SEQ ID NO.5)，从而得到pCDH-MSCV-EGFP-T2A-CD63-MS2-EF1-Puro。Insert the gene synthesized EGFP-T2A-CD63-MS2 (SEQ ID NO.5) into the NheI (3' end) and NotI (5' end) of the backbone vector pCDH-MSCV-MCS-EF1-Puro to obtain pCDH-MSCV -EGFP-T2A-CD63-MS2-EF1-Puro.
2、牙髓干细胞和MSC细胞培养2. Dental pulp stem cells and MSC cell culture
培养基：MSC和牙髓干细胞使用的培养基相同，培养基配方如表1所示。Medium: The medium used for MSC and dental pulp stem cells is the same, and the medium formula is shown in Table 1.
表1 培养基配方Table 1 Medium formula
成分Element 厂家factory 货号Item No.
MEMα培养基MEMα medium 赛默飞世尔科技Thermo Fisher Scientific 1257104812571048
HPL血清HPLserum HycloneHyclone SH30413.02SH30413.02
1％的双抗1% double antibody GibcoGibco 1514016315140163
肝素钠Heparin Sodium sigmasigma H3149-500KU-9H3149-500KU-9
支原体抑制剂Mycoplasma Inhibitors invivoGeninvivoGen ant-mpt-1ant-mpt-1
accutaseaccutase 赛默飞世尔科技Thermo Fisher Scientific 00-4555-5600-4555-56
具体步骤：Specific steps:
①复苏：从液氮罐中取出冻存的MSC和牙髓干细胞，置于37℃水浴中快速解冻，向冻存管中加入1ml MEMα培养基重悬，1300r/min，离心5min，去上清，用MEMα培养基重悬后铺板于10cm细胞培养皿中，37℃，5％的CO 2培养。一个10cm细胞培养皿铺120万细胞，用8ml培养基重悬，间隔一天传代(一般铺80万的时候会间隔2-3天传代)； ①Resuscitation: Take out the frozen MSCs and dental pulp stem cells from the liquid nitrogen tank, place them in a 37°C water bath to thaw quickly, add 1ml MEMα medium to the cryopreservation tube to resuspend, centrifuge at 1300r/min for 5min, and remove the supernatant , resuspended with MEMα medium and plated in a 10cm cell culture dish, cultured at 37°C, 5% CO 2 . Spread 1.2 million cells on a 10cm cell culture dish, resuspend with 8ml of culture medium, and passage at intervals of one day (generally, when laying 800,000 cells, it will pass at intervals of 2-3 days);
②传代：用accutase酶进行消化，每个10cm细胞培养皿中加入3ml的accutase酶，置于37℃消化3min，用3ml的MEMα培养基终止消化，将细胞悬液导入15ml离心管中，1300r/min，离心5min，去上清，用24ml培养基重悬，铺于3个10cm细胞培养皿中37℃，5％的CO 2培养。 ② Passage: Digest with accutase enzyme, add 3ml of accutase enzyme to each 10cm cell culture dish, place at 37°C for 3min, stop digestion with 3ml of MEMα medium, transfer the cell suspension into a 15ml centrifuge tube, 1300r/ min, centrifuge for 5min, remove the supernatant, resuspend with 24ml of culture medium, spread in three 10cm cell culture dishes and culture at 37°C, 5% CO 2 .
3、细胞转染3. Cell transfection
(1)病毒感染前铺板：取两个六孔板，第一个六孔板中每个孔铺30万MSC细胞，第二个六孔板中每个孔铺40万牙髓干细胞。37℃，5％的CO 2培养24h后，用不同滴度的EGFP慢病毒进行感染。 (1) Plating before virus infection: two six-well plates were taken, 300,000 MSC cells were plated in each well of the first six-well plate, and 400,000 dental pulp stem cells were plated in each well of the second six-well plate. After culturing for 24 hours at 37° C. in 5% CO 2 , they were infected with different titers of EGFP lentivirus.
(2)将EGFP慢病毒(滴度为1×10 8vp/ml)三倍稀释4个梯度后，将不同滴度的病毒分别加入5个试验组孔中，留一个孔做对照，每个试验组孔中加入1μl的poly(8μg/μl)，具体加入量如表2所示。 (2) After diluting the EGFP lentivirus (with a titer of 1×10 8 vp/ml) three-fold in 4 gradients, add the viruses with different titers into the wells of the 5 test groups, leaving one well as a control, and each 1 μl of poly (8 μg/μl) was added to the wells of the test group, and the specific addition amount is shown in Table 2.
表2 实验条件Table 2 Experimental conditions
Figure PCTCN2022091281-appb-000005
Figure PCTCN2022091281-appb-000005
4、检测EGFP慢病毒感染一次后、感染三次后和细胞传代三次后的病毒感染效率。4. Detect the virus infection efficiency after EGFP lentivirus infection once, three times after infection and three times after cell passage.
5、检测EGFP在外泌体中的表达5. Detection of EGFP expression in exosomes
使用Cell Culture Media Exosome Purification and RNA Isolation Mini Kit(norgenbiotek，货号：Cat.60700)提取上述细胞上清液中外泌体，并提取总RNA，取适量总RNA反转录，反转录试剂盒为
Figure PCTCN2022091281-appb-000006
II Reverse Transcriptase(全式金，AH101-02)，按照厂家说明书进行反转录。 Use the Cell Culture Media Exosome Purification and RNA Isolation Mini Kit (norgenbiotek, Cat. No.: Cat. 60700) to extract the exosomes in the above cell supernatant, and extract the total RNA, and take an appropriate amount of total RNA for reverse transcription. The reverse transcription kit is
Figure PCTCN2022091281-appb-000006
II Reverse Transcriptase (full gold, AH101-02), reverse transcription was performed according to the manufacturer's instructions.
使用
Figure PCTCN2022091281-appb-000007
PCR SuperMix(全式金，AS111-11)进行PCR，按如表3所述的反应体系进行预混。 use
Figure PCTCN2022091281-appb-000007
PCR SuperMix (whole gold, AS111-11) was used for PCR, and the reaction system described in Table 3 was premixed.
表3 反应体系Table 3 Reaction system
Figure PCTCN2022091281-appb-000008
Figure PCTCN2022091281-appb-000008
Figure PCTCN2022091281-appb-000009
Figure PCTCN2022091281-appb-000009
然后将上述体系放入PCR仪中按照3步法进行反应，循环数为45，反应程序如表4所示。Then the above system was put into a PCR machine to react according to the 3-step method, the number of cycles was 45, and the reaction procedure was shown in Table 4.
表4 反应程序Table 4 Reaction program
Figure PCTCN2022091281-appb-000010
Figure PCTCN2022091281-appb-000010
检测EGFP蛋白的mRNA反转录的cDNA，其中，具体引物的序列如下：Detect the cDNA reverse-transcribed from the mRNA of the EGFP protein, wherein the sequence of the specific primer is as follows:
EGFP qPCR引物序列：EGFP qPCR primer sequences:
正向引物，5'-AAGCTGACCCTGAAGTTCATCTGC-3'(SEQ ID NO.6)；Forward primer, 5'-AAGCTGACCCTGAAGTTCATCTGC-3' (SEQ ID NO.6);
反向引物，5'-CTTGTAGTTGCCGTCGTCCTTGAA-3'(SEQ ID NO.7)。Reverse primer, 5'-CTTGTAGTTGCCGTCGTCCTTGAA-3' (SEQ ID NO.7).
二、结果2. Results
1、不同MOI和转染次数的转染效率检测1. Detection of transfection efficiency with different MOI and transfection times
1)EGFP病毒感染MSC和牙髓干细胞一次后，感染效率如图1和图2、表5和表6所示。1) After the EGFP virus infected MSCs and dental pulp stem cells once, the infection efficiency is shown in Figure 1 and Figure 2, Table 5 and Table 6.
2)EGFP慢病毒感染MSC和牙髓干细胞三次后，感染效率如图3和图4、表5和表6所示。2) After the EGFP lentivirus infected MSCs and dental pulp stem cells three times, the infection efficiency is shown in Figure 3 and Figure 4, Table 5 and Table 6.
表5 EGFP慢病毒感染MSC结果Table 5 Results of EGFP lentivirus infection of MSC
Figure PCTCN2022091281-appb-000011
Figure PCTCN2022091281-appb-000011
表6 EGFP慢病毒感染牙髓干细胞结果Table 6 Results of EGFP lentivirus infection of dental pulp stem cells
Figure PCTCN2022091281-appb-000012
Figure PCTCN2022091281-appb-000012
2、传代后转染效率的检测2. Detection of transfection efficiency after passage
MSC和牙髓干细胞传代三次后，转染效率的改变如图5和图6所示、表7和表8所示。After three passages of MSCs and dental pulp stem cells, the changes in transfection efficiency are shown in Figures 5 and 6, and Tables 7 and 8.
表7 EGFP慢病毒感染传代三次后的MSC细胞Table 7 MSC cells after EGFP lentivirus infection and passage three times
Figure PCTCN2022091281-appb-000013
Figure PCTCN2022091281-appb-000013
表8 EGFP慢病毒感染传代三次后的牙髓干细胞Table 8 Dental pulp stem cells after EGFP lentivirus infection and passage three times
Figure PCTCN2022091281-appb-000014
Figure PCTCN2022091281-appb-000014
Figure PCTCN2022091281-appb-000015
Figure PCTCN2022091281-appb-000015
3、翻译EGFP的mRNA的定位检测3. Localization detection of translation EGFP mRNA
结果如图7显示，翻译EGFP的mRNA出现在牙髓干细胞和MSC分泌的外泌体中。The results are shown in Figure 7, the mRNA translated EGFP appeared in exosomes secreted by dental pulp stem cells and MSCs.
II、干细胞治疗II. Stem cell therapy
一、步骤1. Steps
1、干细胞导入动物体内1. Introducing stem cells into animals
1)把大鼠固定在固鼠器里，把它的尾巴放进盐水瓶里浸泡半分钟至一分钟，然后再用酒精搽尾静脉，使静脉显露出来。1) Fix the rat in the mouse holder, put its tail into the saline bottle and soak for half a minute to one minute, and then rub the tail vein with alcohol to expose the vein.
2)在离尾静脉末端2-3cm出进针，进去后，回抽下看下有没回血，如有回血就可以把要打进去了，如果没回血，就表明没有进静脉，重试。2) Insert and exit the needle at a distance of 2-3cm from the end of the tail vein. After entering, pull back to see if there is any blood return.
3)实验组按照200万细胞/kg体重给药(使用生理盐水配制重悬细胞)，实验组回输相同体积生理盐水。3) The experimental group was administered at the rate of 2 million cells/kg body weight (resuspended cells were prepared with physiological saline), and the experimental group was reinfused with the same volume of normal saline.
4)打完要后，止血，要用消毒的棉签，防止感染。4) After the injection, to stop the bleeding, use a sterile cotton swab to prevent infection.
2、检测EGFP的表达和富集2. Detection of EGFP expression and enrichment
给药24小时后，取主要脏器(心脏、肝脏、脾脏、肺脏、肾脏、脑、睾丸、卵巢、小肠、胃)，提取组织总RNA，qPCR检测脏器中EGFP mRNA表达量。After 24 hours of administration, the main organs (heart, liver, spleen, lung, kidney, brain, testis, ovary, small intestine, stomach) were collected, total RNA was extracted, and the expression of EGFP mRNA in the organs was detected by qPCR.
3、动态监测牙髓干细胞和MSC产生含有EGFP的mRNA的量3. Dynamic monitoring of the amount of EGFP-containing mRNA produced by dental pulp stem cells and MSCs
给药1天、3天、7天、14天、21天后，取主要脏器(心脏、肝脏、脾脏、肺脏、肾脏、脑、睾丸、卵巢、小肠、胃)，提取组织总RNA，qPCR检测脏器中EGFP mRNA表达量。After 1 day, 3 days, 7 days, 14 days, and 21 days of administration, major organs (heart, liver, spleen, lung, kidney, brain, testis, ovary, small intestine, stomach) were collected, and total RNA was extracted for qPCR detection EGFP mRNA expression in organs.
二、结果2. Results
注射牙髓干细胞后，不同时间节点，EGFP的mRNA在动物的各个组织器官表达水平见图8所示。After injection of dental pulp stem cells, the expression levels of EGFP mRNA in various tissues and organs of animals at different time points are shown in Figure 8.
注射间充质干细胞后，不同时间节点，EGFP的mRNA在动物的各个组织器官表达水平见图9所示。After the injection of mesenchymal stem cells, the expression levels of EGFP mRNA in various tissues and organs of the animals at different time points are shown in Fig. 9 .
综上所述，通过慢病毒方式感染自体或异体成熟体细胞或成熟体细胞的前体细胞，达到在外泌体中装载目的治疗基因包括mRNA、miRNA、ncRNA、shRNA、gRNA等。细胞经过一代的培养，然后通过静脉输注到体内。检测发现，导入的基因可以在动物体内不同的组织器官内检测到，同时测定了不同时间节点牙髓干细胞和MSC产生含有EGFP的mRNA的量，证明了以细胞作为载体可以持续有效的向靶细胞递送导入基因。In summary, autologous or allogeneic mature somatic cells or precursor cells of mature somatic cells are infected by lentivirus to load target therapeutic genes in exosomes, including mRNA, miRNA, ncRNA, shRNA, gRNA, etc. The cells are cultured for one generation and then infused intravenously into the body. The test found that the introduced gene can be detected in different tissues and organs in the animal body. At the same time, the amount of EGFP-containing mRNA produced by dental pulp stem cells and MSCs at different time points was measured, which proved that using cells as carriers can continuously and effectively target cells. Delivery of the introduced gene.
实施例2Example 2
I、构建相关载体I. Construct related vectors
本实施例涉及两种载体，一种载体(如图10所示)表达蛋白包括Lamp2b(或PDGFT)，其N端(由BsmBI酶切位点***)可表达包括但不限于识别神经元细胞的RVG蛋白，或识别EGFR受体的GE11蛋白，或其他特定单链抗体等，其C端表达MS2蛋白，用于识别含有acaugaggaucacccaug(SEQ ID NO.8)序列的mRNA；另一种载体则表达含有acaugaggaucacccaug序列的mRNA，以GFP为例，如图11所示，GFP由BsiWI与NheI酶切位点***，可更换为其他目的基因或RNA序列。This embodiment involves two vectors, one vector (as shown in Figure 10) expresses proteins including Lamp2b (or PDGFT), and its N-terminus (inserted by the BsmBI restriction site) can express proteins including but not limited to recognizing neuronal cells RVG protein, or GE11 protein that recognizes EGFR receptors, or other specific single-chain antibodies, etc., its C-terminus expresses MS2 protein, which is used to recognize mRNA containing acaugaggaucacccaug (SEQ ID NO.8) sequence; another vector expresses mRNA containing For the mRNA of acaugaggaucacccaug sequence, take GFP as an example, as shown in Figure 11, GFP is inserted by BsiWI and NheI restriction sites, and can be replaced with other target genes or RNA sequences.
如图10所示为表达外泌体膜蛋白载体pLenti-EF1α-Lamp2b-MS2(核苷酸序列如SEQ ID NO.9所示)，该载体可用于包装慢病毒，并含有puromycin筛选标记，载体含有EF1α启动 子，启动表达Lamp2b-MS2融合蛋白，Lamp2b蛋白N端上膜信号后设计有BsmBI酶切位点，可无缝接入例如识别神经元细胞的RVG蛋白等，载体包含有WPRE原件，可增加相应蛋白表达。As shown in Figure 10, the exosome membrane protein vector pLenti-EF1α-Lamp2b-MS2 (nucleotide sequence shown in SEQ ID NO.9) is expressed. This vector can be used to package lentivirus and contains puromycin selection marker, vector Contains EF1α promoter to promote the expression of Lamp2b-MS2 fusion protein. The N-terminal membrane signal of Lamp2b protein is designed with a BsmBI restriction site, which can be seamlessly connected to, for example, the RVG protein that recognizes neuron cells. The vector contains WPRE components, Can increase the corresponding protein expression.
如图11所示为表达含有MS2识别序列(acaugaggaucacccaug)的目的mRNA载体(以EGFP为例，pLenti-EF1α-EGFP-MS2-Neo载体，核苷酸序列如SEQ ID NO.10所示)，可通过BsiWI及NheI酶切位点连入其他目的基因mRNA序列用于蛋白表达，同样可连入具有功能的功能性miRNA序列，例如：miRNA200cluster(miRNA200、miRNA200b、miRNA200c、miRNA141、miRNA429)，该载体含有NeoR抗性基因，可用G418进行抗性筛选，载体同样可用于包装慢病毒。As shown in Figure 11, it expresses the target mRNA carrier containing the MS2 recognition sequence (acaugaggaucacccaug) (taking EGFP as an example, pLenti-EF1α-EGFP-MS2-Neo carrier, the nucleotide sequence is shown in SEQ ID NO.10), which can be The mRNA sequence of other target genes can be connected into other target gene mRNA sequences for protein expression through BsiWI and NheI restriction sites. It can also be connected into functional miRNA sequences, for example: miRNA200cluster (miRNA200, miRNA200b, miRNA200c, miRNA141, miRNA429), the vector contains NeoR resistance gene, G418 can be used for resistance screening, and the vector can also be used to package lentivirus.
II、构建产生外泌体的工程细胞株II. Construction of engineered cell lines producing exosomes
1、步骤1. Steps
1)以含有识别神经元细胞的RVG蛋白载体(如图12所示，pLenti-EF1α-RVG-Lamp2b-MS2，RVG的核苷酸序列如SEQ ID NO.11所示)包装慢病毒，感染HEK293T细胞，并使用嘌呤霉素进行筛选，获得稳转细胞株(记为293T-RVG-Lamp2b-MS2)。1) Package the lentivirus with the RVG protein vector containing the recognition of neuron cells (as shown in Figure 12, pLenti-EF1α-RVG-Lamp2b-MS2, the nucleotide sequence of RVG is shown in SEQ ID NO.11), and infect HEK293T Cells were screened with puromycin to obtain a stably transfected cell line (referred to as 293T-RVG-Lamp2b-MS2).
2)以含有目的mRNA的载体(如图11所示，pLenti-EF1α-EGFP-MS2-Neo)包装慢病毒，感染293T-RVG-Lamp2b-MS2细胞，使用G418筛选获得稳转细胞株(记为293T-RVG-Lamp2b-MS2-EGFP)。2) Package the lentivirus with a vector containing the target mRNA (as shown in Figure 11, pLenti-EF1α-EGFP-MS2-Neo), infect 293T-RVG-Lamp2b-MS2 cells, and use G418 to screen to obtain a stable cell line (denoted as 293T-RVG-Lamp2b-MS2-EGFP).
3)挑取步骤2)中的单克隆扩大培养293T-RVG-Lamp2b-MS2-EGFP细胞，并收集上清，浓缩外泌体，使用浓缩后的外泌体侵染细胞，观察荧光。3) Pick the monoclonal in step 2) and expand the culture of 293T-RVG-Lamp2b-MS2-EGFP cells, collect the supernatant, concentrate the exosomes, use the concentrated exosomes to infect the cells, and observe the fluorescence.
2、结果2. Results
如图13所示，使用浓缩的外泌体连续三天侵染细胞，可观察到明显的荧光，说明外泌体可将mRNA递送到细胞中。As shown in Figure 13, cells were infected with concentrated exosomes for three consecutive days, and obvious fluorescence was observed, indicating that exosomes can deliver mRNA into cells.
实施例3Example 3
I、GGAG基序对外泌体靶向的影响I. Effect of GGAG motif on exosome targeting
一、miRNA慢病毒表达载体构建1. Construction of miRNA lentiviral expression vector
1、以pLKO.1-puro质粒(sigma，货号SHC001)为miRNA表达的骨架载体，酶切位点是AgeI和EcoRI。1. The pLKO.1-puro plasmid (sigma, Cat. No. SHC001) is used as the backbone vector for miRNA expression, and the restriction sites are AgeI and EcoRI.
2、以渐冻症相关的miRNA(hsa-miR-151a-5p和hsa-miR-155-5p)为例，构建载体：2. Taking miRNAs related to ALS (hsa-miR-151a-5p and hsa-miR-155-5p) as an example, construct the vector:
hsa-miR-151a-5p的序列为UCGAGGAGCUCACAGUCUAGU(SEQ ID NO:12)，hsa-miR-151a-5中存在GGAG序列；The sequence of hsa-miR-151a-5p is UCGAGGAGCUCACAGUCUAGU (SEQ ID NO: 12), GGAG sequence exists in hsa-miR-151a-5;
hsa-miR-155-5p的序列为UUAAUGCUAAUCGUGAUAGGGGUU(SEQ ID NO:13)，hsa-miR-155-5p中不存在GGAG序列。The sequence of hsa-miR-155-5p is UUAAUGCUAAUCGUGAUAGGGGUU (SEQ ID NO: 13), and there is no GGAG sequence in hsa-miR-155-5p.
3、基因合成hsa-miR-151a-5p和hsa-miR-155-5p的shRNA结构，合成公司为安徽通用生物科技有限公司，将hsa-miR-151a-5p/hsa-miR-155-5p的shRNA结构***pLKO.1-puro质粒的AgeI和EcoRI之间，载体命名为pLKO.1-miR-151a-5p-puro和pLKO.1-miR-155-5p-puro(载体示意图如图14和图15所示)；3. Gene synthesis of shRNA structures of hsa-miR-151a-5p and hsa-miR-155-5p, the synthesis company is Anhui General Biotechnology Co., Ltd., hsa-miR-151a-5p/hsa-miR-155-5p The shRNA structure was inserted between AgeI and EcoRI of the pLKO.1-puro plasmid, and the vectors were named pLKO.1-miR-151a-5p-puro and pLKO.1-miR-155-5p-puro (the schematic diagram of the vector is shown in Figure 14 and 15);
hsa-miR-151a-5p的shRNA结构序列为：The shRNA structural sequence of hsa-miR-151a-5p is:
5`-CCGGTCGAGGAGCTCACAGTCTAGTCTCGAGACTAGACTGTGAGCTCCTCGATTTTTG-3`(SEQ ID NO:14)；5`-CCGGTCGAGGAGCTCACAGTCTAGTCTCGAGACTAGACTGTGAGCTCCTCGATTTTTG-3` (SEQ ID NO: 14);
hsa-miR-155-5p的shRNA结构序列为：The shRNA structural sequence of hsa-miR-155-5p is:
5`-CCGGTTAATGCTAATCGTGATAGGGGTTCTCGAGAACCCCTATCACGATTAGCATTAATTTTTG-3`(SEQ ID NO:15)。5`-CCGGTTAATGCTAATCGTGATAGGGGTTCTCGAGAACCCCTATCACGATTAGCATTAATTTTTG-3` (SEQ ID NO: 15).
二、HNRNPA2B1慢病毒表达载体构建2. Construction of HNRNPA2B1 lentiviral expression vector
1、以pLV-EF1a-IRES-Hygro质粒(addgene，货号Plasmid#85134)为HNRNPA2B1表达的骨架载体，酶切位点是EcoRI和Hpa1。1. Use the pLV-EF1a-IRES-Hygro plasmid (addgene, product number Plasmid#85134) as the backbone vector for HNRNPA2B1 expression, and the restriction sites are EcoRI and Hpa1.
2、基因合成HNRNPA2B1序列(其核苷酸序列如SEQ ID NO:16所示，氨基酸序列如SEQ ID NO:17所示)，合成公司为安徽通用生物科技有限公司，将HNRNPA2B1序列***pLV-EF1a-IRES-Hygro质粒的EcoRI和Hpa1之间，载体命名为pLV-EF1a-HNRNPA2B1-IRES-Hygro(载体示意图如图16所示)。2. Gene synthesis of the HNRNPA2B1 sequence (its nucleotide sequence is shown in SEQ ID NO: 16, and its amino acid sequence is shown in SEQ ID NO: 17), the synthesis company is Anhui General Biotechnology Co., Ltd., and the HNRNPA2B1 sequence is inserted into pLV-EF1a - Between EcoRI and Hpa1 of the IRES-Hygro plasmid, the vector is named pLV-EF1a-HNRNPA2B1-IRES-Hygro (the schematic diagram of the vector is shown in Figure 16).
三、慢病毒包装3. Lentiviral packaging
1、细胞接种：10cm dish接种1.5×10 7个293T细胞。加10ml含10％FBS(hyclone，SH30084.03)的DMEM(赛默飞世尔科技，11965084)培养基，37℃，5％CO 2培养箱过夜培养，16-24h后转染； 1. Cell inoculation: inoculate 1.5×10 7 293T cells in a 10cm dish. Add 10ml of DMEM (Thermo Fisher Scientific, 11965084) medium containing 10% FBS (hyclone, SH30084.03), culture overnight at 37°C in a 5% CO 2 incubator, and transfect after 16-24 hours;
2、细胞转染：细胞生长的交汇度达到80-90％，准备转染。转染体系如表9所示。2. Cell transfection: The confluence of cell growth reaches 80-90%, ready for transfection. The transfection system is shown in Table 9.
表9 转染体系Table 9 Transfection system
Figure PCTCN2022091281-appb-000016
Figure PCTCN2022091281-appb-000016
B液逐滴加入A液中，边加边摇匀，室温22-26℃静置15min。逐滴加到培养皿中，轻轻摇匀，5％CO 2，37℃过夜培养。 Add solution B to solution A drop by drop, shake well while adding, and let stand at room temperature 22-26°C for 15 minutes. Add it dropwise to the petri dish, shake gently, and culture overnight at 37°C in 5% CO 2 .
3、转染换液：16-18h后，去掉含转染试剂的培养基，加入10ml含10％FBS的DMEM，5％CO 2，37℃条件下继续培养，(此时开始细胞上清液中将产出病毒)。 3. Transfection medium: After 16-18 hours, remove the medium containing the transfection reagent, add 10ml of DMEM containing 10% FBS, 5% CO 2 , and continue to culture at 37°C (at this time, the cell supernatant will produce virus).
4、病毒第一次收获：从转染开始算48h后，收获细胞上清，转移到50ml离心管中，3,000rpm离心10min，上清用0.45μm滤膜过滤，4℃保存。细胞加入10ml含10％FBS的DMEM，5％CO 2，37℃条件下继续培养。 4. Harvest the virus for the first time: 48 hours after transfection, harvest the cell supernatant, transfer to a 50ml centrifuge tube, centrifuge at 3,000rpm for 10min, filter the supernatant with a 0.45μm filter membrane, and store at 4°C. The cells were added with 10 ml of DMEM containing 10% FBS, 5% CO 2 , and continued to culture at 37°C.
5、病毒第二次收获：收获细胞上清，转移到50ml离心管中，3,000rpm离心10min，上清用0.45μm滤膜过滤，4℃保存。细胞用10％消毒液(84消毒液)处理后丢弃。5. The second virus harvest: Harvest the cell supernatant, transfer it to a 50ml centrifuge tube, centrifuge at 3,000rpm for 10min, filter the supernatant with a 0.45μm filter membrane, and store at 4°C. Cells were discarded after being treated with 10% disinfectant (84 disinfectant).
6、病毒浓缩：将收集到的慢病毒组分用0.45μm滤器过滤去除细菌污染，将过滤后组分与Lenti-XTM Concentrator(clonetech,631232)按照体积比3:1混合，轻轻颠倒混匀。6. Virus concentration: filter the collected lentivirus components with a 0.45 μm filter to remove bacterial contamination, mix the filtered components with Lenti-XTM Concentrator (clonetech, 631232) at a volume ratio of 3:1, and gently invert to mix .
7、4℃孵育30min或过夜。7. Incubate at 4°C for 30 minutes or overnight.
8、4℃1,500g离心45min，离心后会在管底看到白色沉淀。8. Centrifuge at 1,500 g for 45 minutes at 4°C, and a white precipitate will be seen at the bottom of the tube after centrifugation.
9、小心吸去上清液，不能破坏白色沉淀。9. Carefully suck off the supernatant without destroying the white precipitate.
10、用适当体积慢病毒保存液重悬沉淀，并将慢病毒分装、保存于-80℃。10. Resuspend the pellet with an appropriate volume of lentivirus preservation solution, aliquot the lentivirus and store it at -80°C.
四、慢病毒感染间充质干细胞4. Lentivirus infection of mesenchymal stem cells
1、慢病毒转染前18-24小时，将间充质干细胞以1×10 5/cm 2铺到10cm培养皿中。 1. 18-24 hours before lentiviral transfection, spread the mesenchymal stem cells in a 10cm culture dish at 1×10 5 /cm 2 .
2、第二天，用含有8μg/ml polybrene(yeasen，40804ES76)的新鲜培养基替换原培 养基，加入适量病毒悬液。37℃孵育。2. The next day, replace the original medium with a fresh medium containing 8 μg/ml polybrene (yasen, 40804ES76), and add an appropriate amount of virus suspension. Incubate at 37°C.
3、继续培养24小时，用新鲜培养基替换含有病毒的培养基。3. Continue culturing for 24 hours, and replace the medium containing the virus with fresh medium.
4、继续培养。在转染后72-96小时后。添加1μg/ml puromycin(yeasen，60210ES25)和50μg/ml hygromycin(yeasen，60224ES03)筛选阳性细胞。4. Continue to train. After 72-96 hours post transfection. Add 1 μg/ml puromycin (yasen, 60210ES25) and 50 μg/ml hygromycin (yasen, 60224ES03) to screen positive cells.
5、继续培养，收集阳性细胞培养液。5. Continue to cultivate and collect positive cell culture fluid.
6、阳性细胞培养液在2000×g离心30分钟，去除细胞碎片，保存在2-8度。6. Centrifuge the positive cell culture solution at 2000×g for 30 minutes to remove cell debris and store it at 2-8 degrees.
五、外泌体提取检测5. Exosome extraction and detection
1、使用总外泌体分离试剂(赛默飞世尔科技，4478359)分离提取外泌体。1. Use the total exosome isolation reagent (Thermo Fisher Scientific, 4478359) to isolate and extract exosomes.
2、将阳性细胞培养液与总外泌体分离试剂按照2：1比例混合，2-8度静置过夜。2. Mix the positive cell culture medium with the total exosome isolation reagent at a ratio of 2:1, and let stand overnight at 2-8 degrees.
3、第二天将上述混合液在2-8度，以10000g离心力离心2小时。3. The next day, centrifuge the above mixture at 2-8 degrees with a centrifugal force of 10000g for 2 hours.
4、离心后，去除上清液，使用总外泌体分离试剂重悬，得到外泌体，2-8度可以保存1周。4. After centrifugation, remove the supernatant and resuspend with total exosome isolation reagent to obtain exosomes, which can be stored at 2-8 degrees for 1 week.
5、qPCR检测外泌体中miR-151a-5p和miR-155-5p表达水平。5. The expression levels of miR-151a-5p and miR-155-5p in exosomes were detected by qPCR.
六、结果6. Results
结果如图17所示，miR-151a-5p在外泌体中高表达，而miR-155-5p在外泌体中低表达。上述结果表明，GGAG基序影响miRNA靶向外泌体。The results are shown in Figure 17, miR-151a-5p is highly expressed in exosomes, while miR-155-5p is lowly expressed in exosomes. The above results indicated that the GGAG motif affects miRNA targeting to exosomes.
II、基因突变hsa-miR-155-5p改变其外泌体靶向II. Gene mutation hsa-miR-155-5p alters its exosome targeting
一、步骤1. Steps
1、通过基因合成改变hsa-miR-155-5p序列结构，产生以下突变体(表10所示)。1. Alter the hsa-miR-155-5p sequence structure by gene synthesis to produce the following mutants (shown in Table 10).
表10 hsa-miR-155-5p突变体Table 10 hsa-miR-155-5p mutant
野生型Wild type UUAAUGCUAAUCGUGAUAGGGGUU SEQ ID NO:32UUAAUGCUAAUCGUGAUAGGGGUU SEQ ID NO:32
突变体1 Mutant 1 UUAAUGCUAAUCGUGAUAGGAGUU SEQ ID NO:26UUAAUGCUAAUCGUGAUAGGAGUU SEQ ID NO:26
突变体2mutant 2 UUAAUGCUAAUCGUGAUAGGUGUU SEQ ID NO:27UUAAUGCUAAUCGUGAUAGGUGUU SEQ ID NO:27
突变体3mutant 3 UUAAUGCUAAUCGUGAUAGAAGUU SEQ ID NO:28UUAAUGCUAAUCGUGAUAGAAGUU SEQ ID NO:28
突变体4mutant 4 UUAAUGCUAAUCGUGAUAGUUGUU SEQ ID NO:29UUAAUGCUAAUCGUGAUAGUUGUU SEQ ID NO:29
突变体5mutant 5 UUAAUGCUAAUCGUGAUCGGAGUU SEQ ID NO:33UUAAUGCUAAUCGUGAUCGGAGUU SEQ ID NO:33
突变体6mutant 6 UUAAUGCUAAUCGUGAUGGGAGUU SEQ ID NO:34UUAAUGCUAAUCGUGAUGGGAGUU SEQ ID NO:34
突变体7mutant 7 UUAAUGCUAAUCGUGACAGGAGUU SEQ ID NO:30UUAAUGCUAAUCGUGACAGGAGUU SEQ ID NO:30
突变体8mutant 8 UUAAUGCUAAUCGUGAGAGGAGUU SEQ ID NO:31UUAAUGCUAAUCGUGAGAGGAGUU SEQ ID NO:31
2、重复前面步骤，qPCR检测外泌体中miR-155-5p及其突变体表达水平。2. Repeat the previous steps to detect the expression levels of miR-155-5p and its mutants in exosomes by qPCR.
突变体1的shRNA结构序列为：The shRNA structural sequence of mutant 1 is:
5`-CCGGTTAATGCTAATCGTGATAGGAGTTCTCGAGAACUCCUAUCACGAUUAGCAUUAATTTTTG-3`(SEQ ID NO:18)；5`-CCGGTTAATGCTAATCGTGATAGGAGTTCTCGAGAACUCCUAUCACGAUUAGCAUUAATTTTTG-3` (SEQ ID NO: 18);
突变体2的shRNA结构序列为：The shRNA structural sequence of mutant 2 is:
5`-CCGGTTAATGCTAATCGTGATAGGTGTTCTCGAGAACACCUAUCACGAUUAGCAUUAATTTTTG-3`(SEQ ID NO:19)；5`-CCGGTTAATGCTAATCGTGATAGGTGTTCTCGAGAACACCUAUCACGAUUAGCAUUAATTTTTG-3` (SEQ ID NO: 19);
突变体3的shRNA结构序列为：The shRNA structural sequence of mutant 3 is:
5`-CCGGUUAAUGCUAAUCGUGAUAGAAGUUCTCGAGAACUUCUAUCACGAUUAGCAUUAA TTTTTG-3`(SEQ ID NO:25)；5`-CCGGUUAAUGCUAAUCGUGAUAGAAGUUCTCGAGAACUUCUAUCACGAUUAGCAUUAA TTTTTG-3` (SEQ ID NO: 25);
突变体4的shRNA结构序列为：The shRNA structural sequence of mutant 4 is:
5`-CCGGUUAAUGCUAAUCGUGAUAGAAGUUCTCGAGAACUUCUAUCACGAUUAGCAUUAATTTTTG-3`(SEQ ID NO:21)；5`-CCGGUUAAUGCUAAUCGUGAUAGAAGUUCTCGAGAACUUCUAUCACGAUUAGCAUUAATTTTTG-3` (SEQ ID NO: 21);
突变体5的shRNA结构序列为：The shRNA structural sequence of mutant 5 is:
5`-CCGGUUAAUGCUAAUCGUGAUCGGAGUUCTCGAGAACUCCGAUCACGAUUAGCAUUAA TTTTTG-3`(SEQ ID NO:24)；5`-CCGGUUAAUGCUAAUCGUGAUCGGAGUUCTCGAGAACUCCGAUCACGAUUAGCAUUAA TTTTTG-3` (SEQ ID NO: 24);
突变体6的shRNA结构序列为：The shRNA structural sequence of mutant 6 is:
5`-CCGGUUAAUGCUAAUCGUGAUGGGAGUUCTCGAGAACUCCCAUCACGAUUAGCAUUAATTTTTG-3`(SEQ ID NO:20)；5`-CCGGUUAAUGCUAAUCGUGAUGGGAGUUCTCGAGAACUCCCAUCACGAUUAGCAUUAATTTTTG-3` (SEQ ID NO: 20);
突变体7的shRNA结构序列为：The shRNA structural sequence of mutant 7 is:
5`-CCGGUUAAUGCUAAUCGUGACAGGAGUUCTCGAGAACUCCCAUCACGAUUAGCAUUAATTTTTG-3`(SEQ ID NO:22)；5`-CCGGUUAAUGCUAAUCGUGACAGGAGUUCTCGAGAACUCCCAUCACGAUUAGCAUUAATTTTTG-3` (SEQ ID NO: 22);
突变体8的shRNA结构序列为：The shRNA structural sequence of mutant 8 is:
5`-CCGGUUAAUGCUAAUCGUGAGAGGAGUUCTCGAGAACUCCUCUCACGAUUAGCAUUAATTTTTG-3`(SEQ ID NO:23)。5`-CCGGUUAAUGCUAAUCGUGAGAGGAGUUCTCGAGAACUCCUCUCACGAUUAGCAUUAATTTTTG-3` (SEQ ID NO: 23).
二、结果2. Results
结果如图18所示，野生型miR-155-5p、突变体5、突变体6在外泌体中低表达；突变体1，2，3，4，7，8在外泌体中高表达。The results are shown in Figure 18, wild-type miR-155-5p, mutant 5, and mutant 6 were lowly expressed in exosomes; mutants 1, 2, 3, 4, 7, and 8 were highly expressed in exosomes.
尽管已经通过具体实施例描述了本发明，但是常规修改对于本领域技术人员而言将是显而易见的，并且这样的修改旨在落入本发明的范围内。While this invention has been described in terms of specific embodiments, routine modifications will be apparent to those skilled in the art, and such modifications are intended to fall within the scope of this invention.

Claims (50)

  1. 一种细胞疗法，所述细胞疗法包括给有需要者施用可以分泌细胞外囊泡的治疗用的细胞或包含其的细胞治疗剂。A cell therapy comprising administering therapeutic cells capable of secreting extracellular vesicles or a cell therapy agent comprising the same to a person in need.
  2. 根据权利要求1所述的细胞疗法，其特征在于，所述分泌细胞外囊泡的治疗用的细胞来源于植物、微生物、动物、人类。The cell therapy according to claim 1, characterized in that, the therapeutic cells that secrete extracellular vesicles are derived from plants, microorganisms, animals, and humans.
  3. 根据权利要求2所述的细胞疗法，其特征在于，所述分泌细胞外囊泡的治疗用的细胞来源于人类。The cell therapy according to claim 2, characterized in that the therapeutic cells secreting extracellular vesicles are derived from human beings.
  4. 根据权利要求3所述的细胞疗法，其特征在于，所述分泌细胞外囊泡的治疗用的细胞包括自体的、异体的。The cell therapy according to claim 3, characterized in that the therapeutic cells secreting extracellular vesicles include autologous and allogeneic cells.
  5. 根据权利要求1所述的细胞疗法，其特征在于，所述分泌细胞外囊泡的治疗用的细胞包括成熟体细胞或成熟体细胞的前体细胞。The cell therapy according to claim 1, characterized in that the therapeutic cells secreting extracellular vesicles comprise mature somatic cells or precursor cells of mature somatic cells.
  6. 根据权利要求5所述的细胞疗法，其特征在于，所述成熟体细胞的前体细胞包括干细胞。The cell therapy of claim 5, wherein the precursor cells of the mature somatic cells comprise stem cells.
  7. 根据权利要求6所述的细胞疗法，其特征在于，所述干细胞包括全能干细胞、多能干细胞、单能干细胞。The cell therapy according to claim 6, wherein the stem cells include totipotent stem cells, pluripotent stem cells, and unipotent stem cells.
  8. 根据权利要求7所述的细胞疗法，其特征在于，所述多能干细胞包括天然存在的多能干细胞、诱导性多能干细胞。The cell therapy according to claim 7, wherein the pluripotent stem cells include naturally occurring pluripotent stem cells and induced pluripotent stem cells.
  9. 根据权利要求8所述的细胞疗法，其特征在于，所述天然存在的多能干细胞的来源包括神经、皮肤、脂肪、骨髓、脐带、胎盘、骨、脐血、乳牙组织、羊膜、胎盘。The cell therapy according to claim 8, wherein the sources of the naturally occurring pluripotent stem cells include nerve, skin, fat, bone marrow, umbilical cord, placenta, bone, umbilical cord blood, deciduous tooth tissue, amniotic membrane, and placenta.
  10. 根据权利要求9所述的细胞疗法，其特征在于，所述天然存在的多能干细胞包括间充质干细胞、牙髓干细胞、髓鞘前体细胞。The cell therapy according to claim 9, wherein the naturally occurring pluripotent stem cells include mesenchymal stem cells, dental pulp stem cells, and myelin precursor cells.
  11. 根据权利要求1所述的细胞疗法，其特征在于，细胞外囊泡包括外泌体、囊泡、微囊泡、微颗粒、内体来源的囊泡、多囊泡体、凋亡小体及其组合。The cell therapy according to claim 1, wherein the extracellular vesicles include exosomes, vesicles, microvesicles, microparticles, endosome-derived vesicles, multivesicular bodies, apoptotic bodies, and its combination.
  12. 根据权利要求11所述的细胞疗法，其特征在于，细胞外囊泡是外泌体。The cell therapy of claim 11, wherein the extracellular vesicles are exosomes.
  13. 根据权利要求1-12任一项所述的细胞疗法，其特征在于，细胞外囊泡包含治疗有需要者所患疾病的治疗性物质或检测性物质。The cell therapy according to any one of claims 1-12, characterized in that the extracellular vesicles contain a therapeutic substance or a detection substance for treating a disease suffered by a person in need thereof.
  14. 根据权利要求13所述的细胞疗法，其特征在于，所述细胞外囊泡还包括RNA结合多肽的识别基序或RNA结合多肽，所述RNA结合多肽的识别基序引导所述治疗性物质或检测性物质进入细胞外囊泡。The cell therapy according to claim 13, wherein the extracellular vesicle further comprises a recognition motif of an RNA-binding polypeptide or an RNA-binding polypeptide, and the recognition motif of the RNA-binding polypeptide guides the therapeutic substance or The detection substance enters the extracellular vesicles.
  15. 根据权利要求14所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序与所述治疗性物质或检测性物质连接。The cell therapy according to claim 14, wherein the recognition motif of the RNA-binding polypeptide is linked to the therapeutic substance or detection substance.
  16. 根据权利要求15所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序与所述治疗性物质或检测性物质直接连接或间接连接。The cell therapy according to claim 15, characterized in that, the recognition motif of the RNA-binding polypeptide is directly or indirectly linked to the therapeutic substance or detection substance.
  17. 根据权利要求14所述的细胞疗法，其特征在于，所述RNA结合多肽通过所述RNA结合多肽的识别基序识别RNA。The cell therapy of claim 14, wherein the RNA-binding polypeptide recognizes RNA through a recognition motif of the RNA-binding polypeptide.
  18. 根据权利要求17所述的细胞疗法，其特征在于，所述RNA结合多肽包括MS2蛋白、hnRNPA2B1蛋白或细胞外囊泡多肽。The cell therapy according to claim 17, wherein the RNA-binding polypeptide comprises MS2 protein, hnRNPA2B1 protein or extracellular vesicle polypeptide.
  19. 根据权利要求18所述的细胞疗法，其特征在于，所述RNA结合多肽包括细胞外囊泡多肽和MS2蛋白；细胞外囊泡多肽与MS2蛋白连接。The cell therapy according to claim 18, wherein the RNA-binding polypeptide comprises an extracellular vesicle polypeptide and MS2 protein; the extracellular vesicle polypeptide is linked to the MS2 protein.
  20. 根据权利要求19所述的细胞疗法，其特征在于，细胞外囊泡多肽的C端或N端与MS2蛋白连接。The cell therapy according to claim 19, characterized in that, the C-terminal or N-terminal of the extracellular vesicle polypeptide is linked to MS2 protein.
  21. 根据权利要求20所述的细胞疗法，其特征在于，细胞外囊泡多肽的C端与MS2蛋白连接。The cell therapy according to claim 20, characterized in that, the C-terminus of the extracellular vesicle polypeptide is connected to the MS2 protein.
  22. 根据权利要求18所述的细胞疗法，其特征在于，细胞外囊泡多肽包括细胞外囊泡膜蛋白或其片段、细胞外囊泡内的蛋白或其片段。The cell therapy according to claim 18, characterized in that, the extracellular vesicle polypeptide comprises extracellular vesicle membrane protein or fragment thereof, protein in extracellular vesicle or fragment thereof.
  23. 根据权利要求22所述的细胞疗法，其特征在于，所述细胞外囊泡多肽包括以下蛋白或其片段：CD9、CD53、CD63、CD81、CD54、CD50、FLOT1、FLOT2、CD49d、CD71、CD133、CD138、CD235a、ALIX、AARDC1、Syntenin-1、Syntenin-2、Lamp2b、TSPAN8、syndecan-1、syndecan-2、syndecan-3、syndecan-4、TSPAN14、CD37、CD82、CD151、CD231、CD102、NOTCH1、NOTCH2、NOTCH3、NOTCH4、DLL1、DLL4、JAG1、JAG2、CD49d/ITGA4、ITGB5、ITGB6、ITGB7、CD11a、CD11b、CD11c、CD18/ITGB2、CD41、CD49b、CD49c、CD49e、CD51、CD61、CD104、Fc受体、白介素受体、免疫球蛋白、MHC-I或MHC-II成分、CD2、CD3ε、CD3ζ、CD13、CD18、CD19、CD30、CD34、CD36、CD40、CD40L、CD44、CD45、CD45RA、CD47、CD86、CD110、CD111、CD115、CD117、CD125、CD135、CD184、CD200、CD279、CD273、CD274、CD362、COL6A1、AGRN、EGFR、GAPDH、GLUR2、GLUR3、HLA-DM、HSPG2、L1CAM、LAMB1、LAMC1、LFA-1、LGALS3BP、Mac-1α、Mac-1β、MFGE8、SLIT2、STX3、TCRA、TCRB、TCRD、TCRG、VTI1A、VTI1B。The cell therapy according to claim 22, wherein the extracellular vesicle polypeptide comprises the following proteins or fragments thereof: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, AARDC1, Syntenin-1, Syntenin-2, Lamp2b, TSPAN8, syndecan-1, syndecan-2, syndecan-3, syndecan-4, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc CD2, CD3ε, CD3ζ, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86 , CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA -1, LGALS3BP, Mac-1α, Mac-1β, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B.
  24. 根据权利要求14所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序包括以下序列：G-X-Y-G，其中X为G、A、U，Y为A、U。The cell therapy according to claim 14, wherein the recognition motif of the RNA-binding polypeptide comprises the following sequence: G-X-Y-G, wherein X is G, A, U, and Y is A, U.
  25. 根据权利要求24所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序包括以下序列：M-G-X-Y-G，其中，M为A、U；X为G、A、U，Y为A、U。The cell therapy according to claim 24, wherein the recognition motif of the RNA-binding polypeptide comprises the following sequence: M-G-X-Y-G, wherein M is A, U; X is G, A, U, and Y is A, U .
  26. 根据权利要求25所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序包括以下序列：N-M-G-X-Y-G，其中，M为A、U；N为A、C、G、U；X为G、A、U，Y为A、U。The cell therapy according to claim 25, wherein the recognition motif of the RNA-binding polypeptide comprises the following sequence: N-M-G-X-Y-G, wherein M is A, U; N is A, C, G, U; X is G , A, U, Y is A, U.
  27. 根据权利要求26所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序包括以下序列：N-M-G-X-Y-G，其中，M为A、C、G、U；N为A、C、G、U；X为G、A、U，Y为A、U。The cell therapy according to claim 26, wherein the recognition motif of the RNA-binding polypeptide comprises the following sequence: N-M-G-X-Y-G, wherein M is A, C, G, U; N is A, C, G, U ; X is G, A, U, Y is A, U.
  28. 根据权利要求27所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序包括以下序列：L-N-M-G-X-Y-G，其中，M为A、C、G、U；N为A、C、G、U；L为A、C、G、U；X为G、A、U，Y为A、U。The cell therapy according to claim 27, wherein the recognition motif of the RNA-binding polypeptide comprises the following sequence: L-N-M-G-X-Y-G, wherein M is A, C, G, U; N is A, C, G, U ; L is A, C, G, U; X is G, A, U, Y is A, U.
  29. 根据权利要求14所述的细胞疗法，其特征在于，所述RNA结合多肽的识别基序包括SEQ ID NO.8所示的序列。The cell therapy according to claim 14, wherein the recognition motif of the RNA-binding polypeptide comprises the sequence shown in SEQ ID NO.8.
  30. 根据权利要求13所述的细胞疗法，其特征在于，所述治疗性物质改变有需要者体内病变细胞的表型、生理状态。The cell therapy according to claim 13, characterized in that the therapeutic substance changes the phenotype and physiological state of diseased cells in a person in need.
  31. 根据权利要求13所述的细胞疗法，其特征在于，所述治疗性物质包括多核苷酸、蛋白质、多肽、小分子。The cell therapy according to claim 13, characterized in that, the therapeutic substances include polynucleotides, proteins, polypeptides, and small molecules.
  32. 根据权利要求13所述的细胞疗法，其特征在于，多核苷酸包括编码RNA、非编码RNA、DNA。The cell therapy according to claim 13, wherein the polynucleotide comprises coding RNA, non-coding RNA, DNA.
  33. 根据权利要求32所述的细胞疗法，其特征在于，非编码RNA包括ncRNA、shRNA、siRNA、miRNA、gRNA。The cell therapy according to claim 32, wherein the non-coding RNA comprises ncRNA, shRNA, siRNA, miRNA, gRNA.
  34. 根据权利要求31所述的细胞疗法，其特征在于，所述多核苷酸、蛋白质、多肽、小分子是天然存在的细胞外囊泡不含有的。The cell therapy according to claim 31, wherein said polynucleotides, proteins, polypeptides, and small molecules are not contained in naturally occurring extracellular vesicles.
  35. 根据权利要求13所述的细胞疗法，其特征在于，检测性物质包括荧光团。The cell therapy of claim 13, wherein the detection substance comprises a fluorophore.
  36. 根据权利要求13所述的细胞疗法，其特征在于，所述细胞外囊泡还包括靶向多肽，所述靶向多肽识别靶细胞膜蛋白。The cell therapy according to claim 13, characterized in that, the extracellular vesicles further comprise a targeting polypeptide, and the targeting polypeptide recognizes a target cell membrane protein.
  37. 根据权利要求36所述的细胞疗法，其特征在于，所述靶向多肽包括蛋白质、肽、单链抗体或抗体的任何其它衍生物。The cell therapy according to claim 36, wherein the targeting polypeptide comprises a protein, a peptide, a single chain antibody or any other derivative of an antibody.
  38. 根据权利要求36所述的细胞疗法，其特征在于，所述靶向多肽与细胞外囊泡膜蛋白或其跨膜结构域连接。The cell therapy according to claim 36, wherein the targeting polypeptide is linked to an extracellular vesicle membrane protein or its transmembrane domain.
  39. 根据权利要求13-38任一项所述的细胞疗法，其特征在于，所述分泌细胞外囊泡的治疗用的细胞的获取方法包括如下步骤：The cell therapy according to any one of claims 13-38, wherein the method for obtaining therapeutic cells secreting extracellular vesicles comprises the following steps:
    1)将治疗性物质或检测性物质导入细胞外囊泡中；1) introducing therapeutic substances or detection substances into extracellular vesicles;
    2)将步骤1)获得的细胞外囊泡与细胞接触；2) contacting the extracellular vesicles obtained in step 1) with cells;
    3)培养纯化步骤2)的细胞获得包含分泌含有治疗性物质或检测性物质的细胞外囊泡的细胞，获得分泌细胞外囊泡的治疗用的细胞。3) Cultivate and purify the cells in step 2) to obtain cells that secrete extracellular vesicles containing therapeutic substances or detection substances, and obtain therapeutic cells that secrete extracellular vesicles.
  40. 根据权利要求13-38所述的细胞疗法，其特征在于，所述分泌细胞外囊泡的治疗用的细胞的获取方法包括如下步骤：The cell therapy according to claims 13-38, characterized in that the method for obtaining therapeutic cells secreting extracellular vesicles comprises the following steps:
    1)将治疗性物质或检测性物质导入分泌细胞外囊泡的细胞中；1) introducing therapeutic substances or detection substances into cells that secrete extracellular vesicles;
    2)培养纯化步骤1)获得的细胞，获得分泌细胞外囊泡的治疗用的细胞。2) The cells obtained in the purification step 1) are cultured to obtain therapeutic cells secreting extracellular vesicles.
  41. 根据权利要求40所述的细胞疗法，其特征在于，当治疗性物质是RNA多核苷酸时，将其导入分泌细胞外囊泡的细胞的步骤包括；The cell therapy according to claim 40, wherein when the therapeutic substance is RNA polynucleotide, the step of introducing it into cells secreting extracellular vesicles comprises;
    1)将表达包含权利要求24-28中任一项所述的RNA结合多肽识别基序的RNA多核苷酸的载体导入细胞；1) introducing a vector expressing an RNA polynucleotide comprising the RNA-binding polypeptide recognition motif of any one of claims 24-28 into a cell;
    2)将表达hnRNPA2B1或其功能类似物的载体导入细胞；2) introducing the vector expressing hnRNPA2B1 or its functional analog into the cell;
    步骤1)和步骤2)不分先后。Step 1) and step 2) are in no particular order.
  42. 根据权利要求40所述的细胞疗法，其特征在于，当治疗性物质是RNA多核苷酸时，将其导入分泌细胞外囊泡的细胞的步骤包括；The cell therapy according to claim 40, wherein when the therapeutic substance is RNA polynucleotide, the step of introducing it into cells secreting extracellular vesicles comprises;
    1)将表达包含SEQ ID NO.1所示的所述RNA结合多肽识别基序的RNA多核苷酸的载体导入细胞；1) introducing a vector expressing an RNA polynucleotide comprising the RNA-binding polypeptide recognition motif shown in SEQ ID NO.1 into a cell;
    2)将表达权利要求19或20中所述的RNA结合多肽的载体导入细胞；2) introducing the vector expressing the RNA-binding polypeptide described in claim 19 or 20 into the cell;
    步骤1)和步骤2)不分先后。Step 1) and step 2) are in no particular order.
  43. 根据权利要求1所述的细胞疗法，其特征在于，所述细胞治疗剂包含所述治疗用的细胞和药学上允许的载体。The cell therapy according to claim 1, wherein the cell therapy agent comprises the therapeutic cells and a pharmaceutically acceptable carrier.
  44. 根据权利要求43所述的细胞疗法，其特征在于，所述细胞治疗剂为所述治疗用的细胞与生理盐水的混合液。The cell therapy according to claim 43, wherein the cell therapy agent is a mixture of the therapeutic cells and physiological saline.
  45. 根据权利要求43所述的细胞疗法，其特征在于，所述细胞治疗剂为静脉注射剂。The cell therapy according to claim 43, wherein the cell therapy agent is an intravenous injection.
  46. 根据权利要求1所述的细胞疗法，其特征在于，所述细胞治疗剂用量与用药对象的体重正相关。The cell therapy according to claim 1, characterized in that the dosage of the cell therapy agent is positively correlated with the body weight of the subject.
  47. 根据权利要求44所述的细胞疗法，其特征在于，所述治疗剂的用药方法是：The cell therapy according to claim 44, wherein the administration method of the therapeutic agent is:
    (1)将每次治疗量的所述治疗用的细胞溶于100ml的生理盐水中；(1) The cells for the treatment of each treatment dose are dissolved in 100ml of physiological saline;
    (2)将0.1-0.2ml的细胞治疗剂以每分钟60滴的速度静脉注射完毕。(2) Complete the intravenous injection of 0.1-0.2 ml of the cell therapy agent at a rate of 60 drops per minute.
  48. 根据权利要求43-47中任一项所述的细胞疗法，其特征在于，所述细胞治疗剂还包括下述功能性成分中的任意一种或多种：The cell therapy according to any one of claims 43-47, wherein the cell therapy agent further comprises any one or more of the following functional components:
    1)维持所述治疗用的细胞活性的成分；1) A component that maintains the activity of the cells used in the treatment;
    2)促进所述治疗用的细胞增殖的成分。2) A component that promotes cell proliferation for said treatment.
  49. 根据权利要求48所述的细胞疗法，其特征在于，所述功能性成分包括血清替代物、非必需氨基酸、谷氨酰胺、L-丙氨酰-L-谷氨酰胺的稳定化二肽、生长因子及其任意组合。The cell therapy of claim 48, wherein the functional ingredients include serum replacement, non-essential amino acids, glutamine, stabilized dipeptides of L-alanyl-L-glutamine, growth factors and any combination thereof.
  50. 根据权利要求1所述的细胞疗法，其特征在于，所述细胞疗法用于治疗的疾病包括遗传性疾病、肿瘤、自身免疫性疾病、神经***疾病、心血管疾病或胃肠道疾病。The cell therapy according to claim 1, characterized in that the diseases for which the cell therapy is used include genetic diseases, tumors, autoimmune diseases, nervous system diseases, cardiovascular diseases or gastrointestinal diseases.
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