A platform for generating safe cell therapies.

Abstract

To provide therapeutic methods having several benefits compared to cell-based therapeutic agents.SOLUTION: The present specification provides cytoplasts, compositions containing cytoplasts, methods for using cytoplasts, and methods for treating a subject, such as providing a healthy or unhealthy subject with benefits, or treating or diagnosing a disease or condition in a subject. In some embodiments, methods for treating a subject include a step of administering a therapeutically effective amount of a composition containing a cytoplast to a subject. Further, the present specification also provides compositions containing cytoplasts (e.g. pharmaceutical compositions). Further, the present specification also provides kits including manuals for using the compositions or methods.SELECTED DRAWING: None

Description

Technical Field

[0001] Claims of Priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 542,133, filed Aug. 7, 2017. The entire content of the above is incorporated herein by reference.

[0002] Research or Development Supported by the Federal Government This invention was made with government support under Grant No. CA097022 awarded by the National Cancer Institute of the United States. The government has certain rights in this invention.

[0003] Technical Field This disclosure relates, at least in part, to the field of biotechnology, and more particularly, to methods and compositions for using cytoplasts (e.g., enucleated cells) for the treatment, prevention, prophylactic treatment, adjuvant therapy or immunomodulation of diseases in healthy or diseased subjects.

[0004] Sequence Listing[[ID=X]] This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on Aug. 1, 2018 is named Sequence Listing.txt and is 17.1 MB in size.

Background Art

[0005] Current techniques and tools for cell-based therapies often have undesirable and dangerous side effects, such as uncontrolled proliferation, increased mutation rates, and a propensity to elicit anti-DNA immune responses.

Summary of the Invention

[0006] ​​​​​​​This disclosure, at least in part, describes safe and controllable therapeutic agents and / or delivery devices. This method and combination are based on the generation of cytoplasm for use as a material. The product offers several advantages. A method for enucleating cells and generating the cytoplasm, cytoplasm The composition and the method of using the described cytoplasm are compared to conventional cell-based therapeutics. All of these can offer several benefits. Firstly, the cytoplasm and composites described herein can provide several advantages. The product and method may be safer than conventional cell-based therapies. In the application form, for example, genetic material is transferred to a host, including the transfer of nuclear-encoded DNA genes. It can reduce the risk of importation. Further potential safety benefits include: The elephant does not respond to the microenvironment(s), does not proliferate, and (for example, during nucleation) One or more of the following apply: (compared to physocarpous stem cells) they do not contribute to disease progression. It can be done.

[0007] Secondly, the cytoplasm described herein is limited or defined (e.g., known The cells may have a lifespan that is programmable or limited. Thirdly, the cells described herein The cells have a reduced size compared to cells in some other cell-based therapies. It is possible.

[0008] Fourth, the cytoplasm described herein is cryohibernation or cryopreservation. It can maintain its effectiveness later. Cryopreservation involves cooling or freezing, and short-term or long-term storage. The ultra-low temperatures (e.g., -80°C in solid CO2) of biological materials (e.g., cells, cytoplasm) This includes storage at -196°C (e.g., with liquid nitrogen). Freezing hibernation is stored at non-freezing temperatures, e.g., 4°C. This includes short-term cooling and preservation in the suspended animation of biological materials (e.g., cells, cytoplasm). Cryogenic hibernation of the cytoplasm may be advantageous for one or more of the following reasons: Hibernation requires less effort than cryopreservation, and cytoplasmic cells that have undergone cryopreservation can be transported (e.g., by shipping). It is possible to do so.

[0009] Fifth, the cytoplasm described herein can be manipulated in a wide range of ways. For example, cytoplasm The body is manipulated to generate or express the drug itself, or to direct it towards a specific site. It is possible. Other advantages of the present invention that are currently being claimed are described herein.

[0010] Therefore, in this specification, cytoplasm, compositions containing cytoplasm, and methods for using cytoplasm are used. Laws and methods for treating subjects, for example, providing benefits to healthy or unhealthy subjects. Methods of providing, or diseases or conditions in the subject (e.g., cancer or neoplasms, infections) Diseases, inflammatory conditions, neurological disorders (e.g., neurodegenerative diseases), degenerative diseases, autoimmune diseases, cardiovascular diseases ischemic diseases, hereditary or genetic disorders, developmental disorders, ophthalmic diseases, bone diseases, metabolic diseases, Poisoning, idiopathic conditions, or two or more of these, or any disease disclosed herein. A method for treating or diagnosing ) is provided. In some embodiments, the subject is treated The method includes a cytoplasm (e.g., recombinant cytoplasm, any cytoplasm as described herein). The step includes administering a therapeutically effective amount of the composition to the target. In some embodiments, the target The administered cytoplasm can generate therapeutic agents. In some embodiments, the target The cytoplasm administered to the patient contains therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins (e.g.) For example, enzymes, antibodies, antigens, toxins, toxins, cytokines, protein hormones, growth factors, cell surface receptors, or vaccines), therapeutic peptides (e.g., peptide hormones or antigens), small molecule therapeutic agents (e.g., steroids, polyketides, alkaloids, toxins, antibiotics, antiviral drugs, colchicine, taxol, mitomycin, or emtansine), or one or more of therapeutic gene editing factors can be generated. In some embodiments, the cytoplast can be engineered to produce one or more of a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, a therapeutic peptide, a therapeutic small molecule, or a therapeutic gene editing factor. In some embodiments, the cytoplast need not be engineered to produce one or more of a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, a therapeutic peptide, a therapeutic small molecule, or a therapeutic gene editing factor. For example, in some embodiments, one or more of a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, a therapeutic peptide, a small molecule therapeutic agent, or a therapeutic gene editing factor can be produced by the cells from which the cytoplast was obtained.

[0011] In some embodiments, a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, a therapeutic peptide, a small molecule therapeutic agent, or a therapeutic gene editing factor can include a targeting moiety. Non-limiting exemplary targeting moieties that can be produced by or included in the cytoplast include chemokine receptors, adhesion molecules, and antigens.

[0012] In some embodiments, the cytoplasts administered to a subject are a therapeutic DNA molecule, a therapeutic R NA molecules, therapeutic proteins (e.g., enzymes, antibodies, antigens, toxins, cytokines, proteins) Cholesterols, growth factors, cell surface receptors, or vaccines, or currently available (or any therapeutic protein under development), therapeutic peptides (e.g., peptide hormones) (An antigen, or any therapeutic peptide currently available or under development), small Molecular therapeutics (e.g., steroids, polyketides, alkaloids, toxins, antibiotics, antiviral drugs) Drugs such as antacids, analgesics, anticoagulants, antidepressants, anticancer drugs, antiepileptic drugs, antipsychotics, sedatives, Colchicine, Taxol, Mitomycin, Emtansine, or currently available or (This refers to any small molecule therapeutic agent under development), therapeutic gene editing factor, therapeutic nanoparticles, or other Therapeutic agents (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g.) It may contain (oncolytic viruses), exosomes, lipids, or ions. A non-exclusive example of an oncolytic virus is talimodyne laharpalebeck. Talimogene laherparepvec), Onyx-015, GL-ONC1, CV706, Voy Examples include ager-Vl and HSV-1716. Also, several wild-type viruses. For example, vaccinia virus, vesiculitis virus (r stomatitis virus), poliovirus, reovirus, se Senecavirus, ECHO-7, and Semliki Forest Virus (Semliki F) Orest virus also exhibits oncolytic behavior.

[0013] In some embodiments, therapeutic DNA molecules, therapeutic RNA molecules, therapeutic drugs, therapeutic peptides Tides, small molecule therapeutics, or therapeutic gene editing factors are not produced by the cytoplasm. In some embodiments, therapeutic DNA molecules, therapeutic RNA molecules, therapeutic drugs, therapeutic peptides Small molecule therapeutics, or therapeutic gene-editing factors, are packaged within the cytoplasm.

[0014] In some embodiments, DNA molecules, RNA molecules, proteins, peptides, and small molecules are used. Therapeutic drugs and / or gene editing factors are expressed by recombinant DNA. Several embodiments So, cells from which the cytoplasm is derived or obtained contain DNA molecules, RNA molecules, and proteins. , peptides, small molecule therapeutics, and / or gene editing factors, one or more of these It is manipulated to make it so. In some embodiments, the cytoplasm is derived from or obtained Cells produce DNA molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / or This involves manipulating one or more gene editing factors to stably (e.g., permanently) express them. In some embodiments, the cytoplasm of the cell is derived from or obtained from DNA. Molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / or gene editing factors One or more of the offspring are manipulated to transiently express. In some embodiments Cells from which the cytoplasm is derived or obtained are manipulated before enucleation. Several implementations Morphologically, the cytoplasm contains DNA molecules, RNA molecules, proteins, peptides, and small molecule therapeutic agents. , and / or manipulate to transiently express one or more of the gene editing factors (For example, it is performed after enucleation.)

[0015] In some embodiments, DNA molecules, RNA molecules, proteins, peptides, and small molecules are used. Therapeutic drugs and / or gene editing factors affect the cytoplasm of cells from which they originate or are obtained. and are not naturally expressed (i.e., in the absence of manipulation) (i.e., DNA molecules, R NA molecules, proteins, peptides, small molecule therapeutics, and / or gene editing factors are used in cells. (Exogenous to the substance). In some embodiments, DNA molecules, RNA molecules, and tan are used. Proteins, peptides, small molecule therapeutics, and / or gene editing factors are naturally present in the subject. It is not expressed in (i.e., DNA molecules, RNA molecules, proteins, peptides, small molecules) Therapeutic agents and / or gene editing factors are exogenous to the subject. Several implementations In terms of form, DNA molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / or Alternatively, gene editing factors target the desired treatment site (e.g., a tumor or specific tissue) in the target. It is not naturally expressed in organs such as the brain, intestines, lungs, heart, liver, spleen, pancreas, muscles, and eyes. (i.e., DNA molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / Alternatively, gene editing factors are exogenous to the target treatment site.

[0016] In some embodiments, DNA molecules, RNA molecules, proteins, peptides, and small molecules are used. Therapeutic drugs and / or gene editing factors are used within cells from which the cytoplasm originates or is obtained. Naturally (i.e., in the absence of manipulation) expressed (i.e., DNA molecules, RNA molecules, Proteins, peptides, small molecule therapeutics, and / or gene editing factors target the cytoplasm. (and thus innate and endogenous) (for example, the manipulation of cells from which the cytoplasm originates or is obtained) (In the absence of) In some embodiments, DNA molecules, RNA molecules, proteins, peptides Small molecule therapeutics and / or gene editing factors are naturally expressed in the subject. That is, DNA molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / or (The gene editing factor is endogenous to the target.) In some embodiments, DNA Molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / or gene editing factors The child is the target treatment site in the subject (e.g., tumor, or specific tissue, e.g., brain, intestines, It is naturally expressed in the lungs, heart, liver, spleen, pancreas, muscles, eyes, etc. (i.e., DNA components) DNA, RNA molecules, proteins, peptides, small molecule therapeutics, and / or gene editing factors (This is endogenous to the target treatment site.)

[0017] In some embodiments, the therapeutic agent is, for example, a DNA molecule, an RNA molecule, a protein, etc. Pludgeons, small molecule therapeutics, and / or gene editing factors are derived from synthetic cells and cytoplasm. It is loaded into the body.

[0018] In some embodiments, the cytoplasm is compared to the cells from which the cytoplasm is derived or obtained. DNA molecules, RNA molecules, proteins, peptides, small molecule therapeutics, and / or Express modified, truncated, or non-mutant versions and / or copies of gene editing factors. In some embodiments, the cytoplasm is a nucleated cell (e.g., a eukaryotic cell, a mammal). Animal cells (e.g., human cells, or any mammalian cells as described herein), protozoa Cells (e.g., amoeba cells), algal cells, plant cells, fungal cells, invertebrate cells, fish cells It is obtained from cells (amphibian cells, reptile cells, or avian cells).

[0019] In some embodiments, the cytoplasm is of at least two types (e.g., at least two, three, 4, 5 or more different therapeutic DNA molecules, therapeutic RNA molecules, therapeutic tannins Any combination of protein, therapeutic peptide, small molecule therapeutic agent, or therapeutic gene editing factor It can be produced or contained in. For example, in some embodiments, the cytoplasm is It is possible to generate or contain therapeutic DNA molecules and small molecule therapeutic agents. For example, In several embodiments, the cytoplasm generates or contains two different small molecule therapeutic agents. This can be done. For example, in some embodiments, the cytoplasm is a chemokine receptor (e.g., ta It can produce or contain (for targeting) and small molecule therapeutics.

[0020] In some embodiments, the cytoplasm is an immortalized cell, a cancer cell (e.g., any cancer cell) ), obtained from primary (e.g., host-derived) cells, or cell lines. Any non-immortalized cells are obtained from, Immortalization can be achieved using methods known in the art. In some embodiments, The cytoplasm can be obtained from the target's own cells. In some embodiments, the cytoplasm The body can be obtained from cells of the same species as the subject. In some embodiments, the cytoplasmic body is It can be obtained from immune cells. In some embodiments, the cytoplasm is natural killer - (NK) cells, neutrophils, macrophages, lymphocytes, fibroblasts, adult stem cells (for example) hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, mesenchymal stem cells, endothelial stem cells, neural stem cells, olfactory stem cells Somatic stem cells, neural crest stem cells, skin stem cells, or testicular cells), mast cells, basophils, phosphatocytes It is obtained from acidophils or induced pluripotent stem cells.

[0021] In some embodiments, before enucleation, two or more cells (e.g., as disclosed herein) are used. Any of the cells) can be melted by any method disclosed herein or known in the art. They are fused. By enucleating the fusion product, the cytoplasm can be obtained.

[0022] In some embodiments, the first cytoplasm is fused to a cell or a second cytoplasm. In some embodiments, the cells are any nucleated cells (e.g., mammalian cells (e.g., hi) (Atom cells, or any mammalian cells as described herein), protozoan cells (e.g., amoeba cells), Cells, algal cells, plant cells, fungal cells, invertebrate cells, fish cells, amphibian cells, reptile cells These are (animal cells, or avian cells). In some embodiments, the second cell is a synthetic cell. Therefore, the step of fusing a cell with any of the cytoplasms described herein is Methods are provided for altering the behavior of cells, including the fusion of cytoplasmic cells. A method is provided which includes the step of administering a therapeutically effective amount of the selected cells.

[0023] In some embodiments, the second cytoplasm is of the same type as the first cytoplasm. Derived from. In some embodiments, the second cytoplasm is different from the first cytoplasm. It originates from the cells of the p. In some embodiments, the second cytoplasm is at least one therapeutic cell. Therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutic drugs , therapeutic gene editing factors, therapeutic nanoparticles, or contained in the first cytoplasm, if These are therapeutic DNA molecules, therapeutic RNA molecules, and therapeutic DNA molecules expressed by the first cytoplasm. Proteins, therapeutic peptides, small molecule therapeutics, therapeutic gene editing factors, therapeutic nanoparticles and It contains or expresses another therapeutic agent which is the same. In some embodiments, the second cytoplasm , at least one therapeutic DNA molecule, therapeutic RNA molecule, therapeutic protein, therapeutic Plitter, small molecule therapeutic agent, therapeutic gene editing factor, therapeutic nanoparticle, or first cytoplasm A therapeutic DNA molecule contained in or expressed by the first cytoplasm, therapeutic RN A molecule, therapeutic protein, therapeutic peptide, small molecule therapeutic agent, therapeutic gene editing factor, therapeutic It contains or expresses a therapeutic agent different from the therapeutic nanoparticles. In some embodiments, The cytoplasm of 1 is subjected to any method known in the art, for example, a virus-based cell surface patch. Using electrofusion or viral fusion with plutidops, a cell or a second cytoplasm is created in the cell or a second cytoplasm. It can be fused together.

[0024] In some embodiments, the therapeutic RNA molecule is messenger RNA (mRNA), Short hairpin RNA (shRNA), small interfering RNA (siRNA), microR NA, long non-coding RNA (IncRNA), or RNA virus. In the embodiment, the therapeutic DNA molecule is single-stranded DNA, double-stranded DNA, oligonucleotides. These are plasmids, bacterial DNA molecules, or DNA viruses. In some embodiments, they are plasmids, bacterial DNA molecules, or DNA viruses. Therapeutic proteins are based on cytokines, growth factors, hormones, antibodies, and small peptides. It is a drug or an enzyme. In some embodiments, the cytoplasm is a therapeutic DNA molecule, therapeutic Therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutics, and / or therapeutics A therapeutic gene editing factor is transiently expressed. In some embodiments, a therapeutic DNA molecule is used. Therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutics, and / or The expression of therapeutic gene editing factors is inducible. In some embodiments, nucleated cells are used for therapeutic purposes. Therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutic drugs , and / or permanently manipulated to express therapeutic gene editing factors. In this embodiment, therapeutic DNA molecule, therapeutic RNA molecule, therapeutic protein, therapeutic peptide Expression of cytotoxic drugs, small molecule therapeutic agents, and / or therapeutic gene editing factors. As described herein. In some embodiments of the method, the cytoplasm contains a therapeutic agent or nanoparticles. In some embodiments, the therapeutic agent is a small molecule, a bacterium, or an exosome.

[0025] In some embodiments, the method involves steps applied to one or more additional therapies. The following further includes: In some embodiments, one or more additional therapies are cell-based. Therapies ranging from small molecule therapy, immunotherapy, chemotherapy, radiation therapy, gene therapy, and surgery. Selected from the group.

[0026] This specification refers to therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides. Tide, small molecule therapeutics, therapeutic gene editing factors, therapeutic nanoparticles, and / or other therapeutics Isolated cytoplasm containing the agent (e.g., recombinant cytoplasm, or any of the agents described herein) A cytoplasm is provided. In some embodiments, the therapeutic agent is a drug or chemotherapy agent. These are gene editing agents.

[0027] Furthermore, this specification describes how to create recombinant cytoplasm (for example, any cytoplasm described herein). A method for producing a therapeutic DNA molecule and a therapeutic RNA molecule, comprising the steps of enucleating nucleated cells and preparing therapeutic DNA molecules and therapeutic RNA molecules. Molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutics, therapeutic gene editing factors, treatment A method comprising the step of introducing nanoparticles and / or another therapeutic agent into enucleated cells, Provided. In some embodiments, the introduction step precedes the denucleation step. In some embodiments, the denucleation step precedes the introduction step. In several embodiments, the introduction step involves a therapeutic DNA molecule, a therapeutic RNA molecule, and a therapeutic agent. Proteins for therapeutic use, peptides for therapeutic use, small molecule therapeutic drugs, gene editing factors for therapeutic use, or other therapeutic drugs This results in the transient expression of the agent. In some embodiments, (for example, the introduction step is If the enucleation step is preceded, the introduction step involves a therapeutic DNA molecule and a therapeutic R NA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutics, therapeutic gene editing factors, Or it leads to the persistent expression of other therapeutic agents. In some embodiments, therapeutic RNA molecules These include messenger RNA (mRNA), short hairpin RNA (shRNA), and low-molecular-weight RNA. Interfering RNA (siRNA), microRNA, long non-coding RNA (IncRNA), or RNA viruses. In some embodiments, the therapeutic DNA molecule is a single-stranded DNA. NA, double-stranded DNA, oligonucleotide, plasmid, bacterial DNA molecule, or DNA It is a virus. In some embodiments, therapeutic DNA or therapeutic RNA is a gene. It is a therapy. In some embodiments, the therapeutic protein is an enzyme, antibody, toxin, site These are kine, protein hormones, growth factors, or vaccines. In some embodiments, Nucleated cells can survive under various conditions (for example, in a cytokine bath or under hypoxic conditions). Before enucleation, (for example, in suspension, as adherent cells, 3D (for example, semi-suspension method or other) They can be cultured as adherent cells in non-adherent methods.

[0028] Furthermore, this specification describes the steps of transfecting nucleated cells with a vector; A method for producing recombinant cytoplasm is also provided, which includes the step of enucleating fect cells. .

[0029] In some embodiments, the vector is a viral vector (e.g., a retroviral vector) TER (e.g., lentiviral vector), adeno-associated virus (AAV) vector, vesicle Viral vectors (e.g., vesicular stomatitis virus (VSV) vectors), or hives It is a lid virus vector. In some embodiments, the viral vector is used in cells It may be a nuclear replicating virus. In some embodiments, the viral vector is a nuclear replicating virus. It may be a case of enucleation. In some embodiments, enucleation occurs when the vector is incorporated into the genome of a nucleated cell. This is done after the cells have been enucleated. In some embodiments, the vector is used after the cells have been enucleated. Transfection is performed. The order of transfection and enucleation affects the choice of vector. It may have an effect. For example, if transfection is performed before enucleation, the cytoplasmic compound Either a synthetic virus or a nuclear-replicating virus can be an acceptable vector. For example, If transfection is performed after enucleation, cytoplasmic replicating viruses will be more effective than nuclear replicating viruses. This could be a good vector choice. On the other hand, nuclear replicating viruses enter the enucleated cytoplasm. It can be packaged and released when the cytoplasm dies. In some embodiments, The ctor contains the coding sequence for a therapeutic protein. In some embodiments, the therapeutic protein Proteins are enzymes, antibodies, toxins, cytokines, protein hormones, growth factors, or other proteins. It's Kuchin.

[0030] Furthermore, this specification refers to therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic Includes cytoplasm expressing peptides, small molecule therapeutics, and / or therapeutic gene editing factors. A method for treating a subject is also provided, which includes the step of administering a therapeutically effective amount of the composition to the subject. In some embodiments, this method involves therapeutic DNA molecules, therapeutic RNA molecules, therapeutic Developing proteins, therapeutic peptides, small molecule therapeutics, and / or therapeutic gene editing factors. Targeting therapeutically effective amounts of compositions containing naturally derived or manipulated cytoplasm. This may include a step of administration.

[0031] Furthermore, this specification refers to therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic Peptides, small molecule therapeutics, therapeutic gene editing factors, therapeutic nanoparticles, and / or other The step includes administering a therapeutically effective amount of a composition containing a cytoplasm containing a therapeutic agent to a target. Methods for treating the target will also be provided.

[0032] In some embodiments, the cytoplasm is obtained from mammalian cells. In this context, the cytoplasm is obtained from immune cells.

[0033] In some embodiments, the composition further includes a targeting portion. In terms of application morphology, the targeting portion is a cell surface protein.

[0034] In some embodiments, the targeting portion is a secreted protein or an extracellular target It is a protein tethered to Rix.

[0035] Unless otherwise defined, all technical and scientific terms used herein are defined in this text. This invention has the same meaning as that generally understood by those skilled in the art in which the invention pertains. Methods and materials for use in this context are described herein. Other methods and materials known in the art are also described herein. Appropriate methods and materials may also be used. Materials, methods, and examples are merely illustrative. This is an indication and not intended to limit all of the references made herein. Publications, patent applications, patents, sequences, database entries, and other references are subject to the law. The entire text is incorporated by reference. In case of any conflict, this specification, including its definitions, shall prevail.

[0036] Other features and advantages of the present invention are described in detail below and in the drawings, as well as in the claims. This should be clear from the scope. [Brief explanation of the drawing]

[0037] [Figure 1] Figure 1 is a schematic diagram of one embodiment of the present disclosure in which the starting material is either donor-derived allogeneic or autologous cells, or engineered cells having one or more designed functions. [Figure 2]Figure 2A is a representative fluorescence microscopy image of cytoplasm generated from human telomerase reverse transcriptase (hTERT) adipose-derived human mesenchymal stem cells (MSCs). Cells were stained with the red dye (5-(and-6)-((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) (CMTMR), and the nuclei were stained with Vybrant® Dyecycle® green. Arrows point to normal nucleated cells, and arrowheads point to enucleated cytoplasm. Scale bar = 50 μm. Figure 2B is a representative fluorescence microscopy image of cytoplasm generated from HL-60 human neutrophil cells (neutrophils). Cells were stained with the red dye CMTMR, and the nuclei were stained with Vybrant® Dyecycle® green. Arrows point to nucleated cells, and arrowheads point to enucleated cytoplasm. Scale bar = 50 μm. Figure 2C is a representative fluorescence microscopy image of cytoplasm generated from NIH3T3 mouse fibroblasts (fibroblasts). Cells were stained with the red dye CMTMR, and the nuclei were stained with Vybrant® Dyecycle® green. Arrows indicate nucleated cells, and arrowheads indicate enucleated cytoplasm. Scale bar = 50 μm. Figure 2D is a representative fluorescence microscopy image of cytoplasm generated from the human natural killer cell line NKL cells (NK). Cells were stained with the green dye 5-chloromethylfluorescein diacetate (CMFDA), and the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Arrows indicate nucleated cells, and arrowheads indicate enucleated cytoplasm. Scale bar = 50 μm. [Figure 3-1] Figure 3A is a representative graph showing the relative rate of change in living cells or cytoplasm over time. [Figure 3-2] Figure 3B is a representative graph showing viable cells and cytoplasm after recovery from cryopreservation. Figure 3C is a representative graph showing the relative viability of cytoplasm 24 hours after enucleation (fresh cytoplasm) or 24 hours after recovery from cryopreservation following enucleation, comparing the viability of fresh cytoplasm and cryopreserved cytoplasm with that of cytoplasm 4 hours after enucleation. Mean ± SEM; n=10. [Figure 4]Figure 4 is a representative flow cytometry graph showing the number of events counted relative to the signal intensity of the indicated fluorescent antibodies / markers (CD90, CD44, CD146, CD166, CD45, and isotype control). Bone marrow MSCs or MSC-derived cytoplasm were stained 24 hours after enucleation and then analyzed by flow cytometry using FlowJo software. Green (light gray) represents nucleated MSCs, and red (dark gray) represents enucleated cytoplasm. [Figure 5]Figure 5A is a representative confocal microscope image of an MSC-derived cytoplasm cultured in a two-dimensional (2D) glass-bottom chamber, stained with rhodamine phalloidin to visualize the F-actin cytoskeleton, and stained with DAPI to visualize the nucleus. The arrows point to the stained cytoskeletal structures. Scale bar = 50 μm. Figure 5B is a representative confocal microscope image of an MSC cultured in a 2D glass-bottom chamber, stained with rhodamine phalloidin to visualize the F-actin cytoskeleton, and stained with DAPI to visualize the nucleus. The arrows point to the stained cytoskeletal structures. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 5C is a representative confocal microscope image of an MSC-derived cytoplasm cultured in a 2D glass-bottom chamber, stained with anti-α-tubulin antibody to visualize the microtubule network, and stained with DAPI to visualize the nucleus. The arrows point to the stained cytoskeletal structures. Scale bar = 50 μm. Figure 5D is a representative confocal microscope image of an MSC cultured in a 2D glass-bottom chamber, stained with anti-α-tubulin antibody to visualize the microtubule network, and stained with DAPI to visualize the nucleus. The arrows point to the stained cytoskeletal structures. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 5E is a representative confocal microscope image of an MSC-derived cytoplasm cultured for 24 hours in a 3D collagen matrix, stained with rhodamine phalloidin to visualize the F-actin cytoskeleton, and stained with DAPI to visualize the nucleus. Scale bar = 50 μm. Figure 5E' is a representative confocal microscope image of 5E showing unmerged DAPI staining for nucleus visualization. Scale bar = 50 μm. Figure 5F is a representative confocal microscope image of an MSC cultured for 24 hours in a 3D collagen matrix, stained with rhodamine phalloidin to visualize the F-actin cytoskeleton, and stained with DAPI to visualize the nucleus. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 5F' is a representative confocal microscope image of 5F showing unbound DAPI staining for nucleus visualization. Scale bar = 50 μm. [Figure 6]Figure 6A is a representative phase-contrast microscope image of MSCs cultured in complete medium for 16 hours, then fixed and stained with crystal violet. The arrowheads point to very clear nanotubes. Scale bar = 50 μm. Figure 6B is a representative confocal microscope image of MSCs cultured in complete medium for 16 hours, then fixed and stained with the mitochondrial marker anti-apoptosis-inducing factor (AIF) and DAPI to visualize the nucleus. The arrowheads point to very clear nanotubes due to protruding mitochondrial staining. Green (light gray) represents AIF-labeled mitochondria. Blue (dark gray) represents DAPI-stained nuclei. Scale bar = 50 μm. Figure 6C is a representative phase-contrast microscope image of MSC-derived cytoplasm cultured in complete medium for 16 hours, then fixed and stained with crystal violet. The arrowheads point to very clear nanotubes. Scale bar = 50 μm. Figure 6C' is a magnified image of Figure 6C'. The arrowheads point to very clear nanotubes. Scale bar = 50 μm. Figure 6D is a representative confocal microscope image of an MSC-derived cytoplasm, cultured in complete medium for 16 hours, then fixed, stained with mitochondrial markers anti-AIF and DAPI, and with the nucleus visualized. The arrowheads point to very clear nanotubes due to protruding mitochondrial staining. Green (light gray) represents AIF-labeled mitochondria. Blue (dark gray) represents DAPI-stained nuclei. Scale bar = 50 μm. Figure 6D' is a magnified image of Figure 6D. Scale bar = 50 μm. [Figure 7]Figure 7A is a representative confocal microscopy image of adipose-derived MSCs stained with the mitochondrial markers anti-AIF (light gray) and DAPI (dark gray oval). The arrows point to mitochondria. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 7A' is a representative confocal microscopy image of an MSC-derived cytoplasm stained with the mitochondrial markers anti-AIF (light gray) and DAPI (dark gray oval). The arrows point to mitochondria. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 7B is a representative confocal microscopy image of adipose-derived MSCs stained with the lysosomal markers anti-lysosome-associated membrane protein 1 (LAMP1, light gray) and DAPI (dark gray oval). The arrows point to lysosomes. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 7B' is a representative confocal microscopy image of an MSC-derived cytoplasm stained with LAMP1 (light gray) and DAPI (dark gray oval). The arrows point to lysosomes. The arrowhead points to the nucleus. Scale bar = 50 μm. Figure 7C is a representative confocal microscopy image of adipose-derived MSCs stained with Golgi marker anti-receptor-binding cancer antigens expressed in SiSo cells (RCAS1, light gray) and DAPI (dark gray oval). The arrow points to the Golgi. The arrowhead points to the nucleus. Scale bar = 50 μm. Figure 7C' is a representative confocal microscopy image of MSC-derived cytoplasm stained with Golgi marker anti-RCAS1 (light gray) and DAPI (dark gray oval). The arrow points to the Golgi. The arrowhead points to the nucleus. Scale bar = 50 μm. Figure 7D is a representative confocal microscopy image of adipose-derived MSCs stained with endoplasmic reticulum (ER) marker anti-protein disulfide isomerase (PDI, light gray) and DAPI (dark gray oval). The arrow points to the ER. The arrowhead points to the nucleus. Scale bar = 50 μm. Figure 7D' is a representative confocal microscopy image of MSC-derived cytoplasm stained with the endoplasmic reticulum (ER) markers anti-PDI (light gray) and DAPI (dark gray oval). The arrows point to the ER. The arrowheads point to the nucleus. Scale bar = 50 μm. Figure 7E is a representative confocal microscopy image of adipose-derived MSCs stained with the endosomal markers anti-early endosomal antigen 1 (EEA1, light gray) and DAPI (dark gray oval). The arrows point to lysosomes. The arrowheads point to the nucleus.Scale bar = 50 μm. Figure 7E' shows representative confocal microscopy images of MSC-derived cytoplasm stained with the endosomal markers anti-EEA1 (light gray) and DAPI (dark gray oval). Arrows point to lysosomes. Arrowheads point to the nucleus. Scale bar = 50 μm. [Figure 8] Figure 8A shows representative bright-field microscopy images of MSCs or cytoplasm in a Boyden chamber assay, successfully migrated to the bottom of an 8.0 μm porous filter over 3 hours and then stained with crystal violet. In the negative control, cells and cytoplasm were migrated in culture medium containing 2% FBS in both the upper and lower chambers. In the stimulated group, the bottom of the upper chamber was coated with fibronectin, and 100 ng / mL of stromal cell-derived factor 1-alpha (SDF-Iα) was added to the lower chamber. Scale bar = 50 μm. Figure 8B is a representative bar graph showing the ratio of migrating MSCs or cytoplasm treated as in Figure 8A (negative or stimulated), with each amount normalized against the loading control (MSCs or cytoplasm directly attached to the fibronectin-coated plate). Mean ± SEM; n=10. [Figure 9]Figure 9A is a representative epifluorescence microscopy image of MSCs incubated with 100 μM cell-permeable peptide (Arg)9-FAM (fluorescein amidite, light gray) and stained with Hoechst 33342 (nucleus, dark gray oval). The arrows indicate Hoechst-stained nuclei. The arrowheads indicate a positive (Arg)9-FAM signal. Figure 9B is a representative epifluorescence microscopy image of cytoplasm derived from MSCs incubated with 100 μM cell-permeable fluorescent peptide (Arg)9-FM (light gray) and stained with Hoechst 33342 (nucleus, dark gray oval). The arrows indicate Hoechst-stained nuclei. The arrowheads indicate a positive (Arg)9-FAM fluorescence. Figure 9C is a representative epifluorescence microscopy image of MSCs incubated with 100 μM doxorubicin (light gray) and stained with Hoechst 33342 (slightly dark gray oval). The arrows indicate Hoechst-stained nuclei. Figure 9D is a representative epifluorescence microscopy image of MSC-derived cytoplasm incubated with 100 μM doxorubicin (light gray) and stained with Hoechst 33342 (slightly dark gray oval). The arrows indicate positive doxorubicin fluorescence. Figure 9E is a representative bar graph showing the mean corrected total cell fluorescence per unit area of ​​cells and cytoplasm, modeling relative fluorescence while considering the size difference between cells and cytoplasm. Corrected total cell fluorescence = integrated density - (area of ​​selected cells * mean fluorescence of background measurements). Mean ± SEM; n=10. [Figure 10]Figure 10A is a panel of merged and unmerged confocal microscopy and phase-contrast images of MSCs and MSC-derived cytoplasm after incubation for 24 hours with small interfering RNA (siRNA) labeled with fluorescein isothiocyanate (FITC, light gray dots) and staining with Hoechst 33342 (nuclei, neutral gray ovals). Arrowheads indicate positive FITC-labeled siRNA fluorescence. Arrows indicate nuclei. Scale bar = 50 μm. Figure 10B is a representative bar graph showing the mean-corrected total cell fluorescence of cells and cytoplasm, modeling relative fluorescence while considering the size difference between cells and cytoplasm. Corrected total cell fluorescence = combined density - (area of ​​selected cells * mean fluorescence of background measurements). [Figure 11-1] Figure 11A shows representative merged and non-merged epifluorescence microscopy images of MSCs and MSC-derived cytoplasm 20 hours after transfection with purified enhanced green fluorescent protein messenger RNA (EGFP-mRNA), stained with Hoechst 33342. [Figure 11-2] Figure 11B is a representative bar graph showing the EGFP mRNA transfection efficiency (percentage of transfected cells out of total cells) of MSCs or MSC-derived cytoplasms treated as in Figure 11A. Mean ± SEM; n=3. Figure 11C is a representative bar graph showing the relative EGFP fluorescence intensity between cells and cytoplasms, illustrating the size differences. Mean ± SEM; MSC group, n=27; MSC-derived cytoplasm group, n=23. Corrected total cell fluorescence = combined density - (area of ​​selected cells * mean fluorescence of background measurements). All data are representative of at least two independent experiments. [Figure 12-1]Figure 12A shows representative Western blots of cells treated for 10 minutes with either control medium, MSC-conditioned medium (MSC CM), MSC-derived cytoplasm-conditioned medium (cytoplasm CM), or 50 ng / mL vascular endothelial growth factor (VEGF). Immunoblotting was performed for protein kinase B (Akt), phosphorylated Akt (p-Akt), extracellular signal-regulated kinase (Erk), and phosphorylated Erk (p-Erk). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Figure 12B is a representative bar graph showing the relative gaussiacalciferase (Glue) activity in the culture medium 48 hours after plating of MSCs transfected with Glue mRNA (MSC-Gluc), untransfected MSC control cells (MSCs), cytoplasm derived from MSCs transfected with Glue mRNA (cytoplasm-Gluc), and cytoplasm derived from untransfected MSCs (cytoplasm). [Figure 12-2] Figure 12C is a representative bar graph showing the percentage of RAW macrophages migrating towards the indicated gradient of conditioned medium over 4 hours in a Boyden chamber assay. Migrating cells on the lower membrane surface were stained with crystal violet, and cell counts were counted per field and normalized to a loading control (cells directly attached to a fibronectin-coated plate). Colony-stimulating factor 1 (CSF-1), 40 ng / mL mouse CSF-1 as positive control; MSC medium, conditioned medium from MSCs; cytoplasmic medium, conditioned medium from MSC-derived cytoplasm. Mean ± SEM; n=10. [Figure 13-1]Figure 13A is a schematic diagram of interleukin-10 (IL-10) mRNA transfected into MSCs and cytoplasm. The Kozak sequence was added before the start codon of the IL-10 mRNA coding region (CDS). The 5'UTR and 3'UTR of human betaglobin (HBB) mRNA were added to the 5' and 3' ends of the IL-10 CDS, respectively. An artificial 5' cap was added to the 5' end of the IL-10 mRNA to manipulate pseudouridine modification and enhance mRNA stability. Figure 13B is a bar graph showing IL-10 concentrations in culture medium of transfected (++) or untransfected (-) MSCs or MSC-derived cytoplasm. MSC-derived cytoplasm was transfected with IL-10 mRNA and then seeded in 24-well plates at 2.5 × 10⁴ cells / well. The conditioned medium (CM) was collected 24 hours after transfection, and the IL-10 concentration was determined by ELISA. [Figure 13-2] Figure 13C is an immunoblot showing the protein expression of Stat3 and phosphorylated Stat3 (P-Stat3, a marker of IL-10 activation) in serum-starved RAW macrophage cells treated for 1 hour with the conditioned medium (CM) shown from MSCs or cytoplasm, as treated as in Figure 13B. Untreated = control without CM treatment. Complete medium = RAW cells treated in MSC complete culture medium. MSC Ctrl = RAW cells treated with CM from untransfected MSCs. MSC IL-10 = RAW cells treated with CM from MSCs transfected with IL-10 mRNA. Cytoplasmic Ctrl = RAW cells treated with CM from untransfected cytoplasm. Cytoplasmic IL-10 = RAW cells treated with CM from cytoplasm transfected with IL-10 mRNA. Figure 13D is a bar graph showing the concentration of secreted IL-10 cytokine in mouse blood determined by ELISA. MSCs or cytoplasm derived from MSCs were processed as shown in Figure 13B and injected into the posterior orbital vascular system of C57BL / 6 mice. Two hours after injection, the animals were euthanized, and blood samples were collected by cardiac puncture. Mean ± SEM; n=3. [Figure 14]Figure 14A is a representative bright-field microscopy image of crystal violet-stained MSCs or MSC-derived cytoplasm in a Boyden chamber assay, which penetrated the underside of an 8.0 μm porous filter coated with basement membrane extract (BME) towards 10% FBS as a chemoattractant for 24 hours. Negative = no FBS (negative control). Scale bar = 50 μm. Figure 14B is a representative bar graph showing the ratio of MSCs or MSC-derived cytoplasm treated as in Figure 14A that penetrated the underside of the membrane compared to a loading control. Mean ± SEM; n=18. [Figure 15-1] Figure 15A shows representative epifluorescence (upper panel) and phase-contrast (lower panel) images of MSCs and cytoplasm in suspension medium. The actin cortex was stained with Lifeact RFP, and the cell nucleus was stained with Vybrant® Dyecycle® green. Arrows point to the cytoplasm, and arrowheads point to the MSC nucleus. Scale bar = 20 μm. Figure 15B is a representative scatter plot showing the size distribution of MSCs and cytoplasm measured with Nikon Element software. Mean ± SEM; n=80. [Figure 15-2] Figure 15C is a representative bar graph showing the detected Vybrant® DiD-labeled MSCs or cytoplasm in the lung. MSCs or cytoplasm were labeled with a DiD dye and injected retroorbitally into the vascular system of C57BL / 6 mice. Tissue was collected 24 hours later, and the cell suspension was analyzed by flow cytometry. Mean ± SEM; n=3. Figure 15D is a representative bar graph showing the detected Vybrant® DiD-labeled MSCs or cytoplasm in the liver. Mean ± SEM; n=3. MSCs or cytoplasm were labeled with a DiD dye and injected retroorbitally into the vascular system of C57BL / 6 mice. Tissue was collected 24 hours later, and the cell suspension was analyzed by flow cytometry. [Figure 16-1] Figure 16A is a schematic diagram of representative lentiviral vectors engineered to express CXCR4 in MSCs and cytoplasm (SEQ ID NOs: 1-15). [Figure 16-2]Figure 16B is a representative flow cytometry graph showing the number of events counted relative to the signal intensity of cell surface CXCR4 expression by fluorescent antibody in engineered cytoplasm and engineered parental MSCs, as analyzed by Flow Jo. Figure 16C is a representative bar graph showing the ratio of migrating cells or cytoplasm that migrated to the underside of the Boyden chamber membrane compared to the loading control. Mean ± SEM; n=10. Engineered MSCs with and without the CXCR4 receptor, as shown in Figure 16A, and MSC-derived cytoplasm were able to migrate toward the indicated concentrations of SDF-Iα for 2 hours in the Boyden chamber assay. [Figure 17-1] Figure 17A is a schematic diagram of a lentiviral vector engineered to express PSGL1 (P-selectin glycoprotein ligand 1) and Fut7 (fucosyltransferase, glycosylate / PSGL1 activator) in MSCs and cytoplasm. The coding sequences of PSGL1 (SEQ ID NO: 16) and Fut7 (SEQ ID NO: 17) were ligated by the 2A sequence (SEQ ID NO: 18). [Figure 17-2] Figure 17B is a representative flow cytometry graph showing the number of events counted relative to the signal intensity of cell surface PSGL1 expression by fluorescent antibody in engineered cytoplasm and engineered parental MSCs, as analyzed by Flow Jo. Figure 17C is a representative graph showing cell surface binding of P-selectin to engineered MSCs and MSC-derived cytoplasm, as determined by flow cytometry. MSC control = parental MSC. Engineered MSC = PSGL1 / Fut7 engineered MSC. Engineered cytoplasm = PSGL1 / Fut7 engineered MSC-derived cytoplasm. [Figure 18-1] Figure 18A is a schematic diagram of a lentiviral vector engineered to express mCD47 (SEQ ID NO: 19) in MSCs and cytoplasm. [Figure 18-2] Figure 18B is a representative flow cytometry graph showing the number of events counted against the cell surface signal intensity of mCD47 expression in manipulated cytoplasm and MSCs, as analyzed by Flow Jo. [Figure 18-3] Figure 18C is a representative bar graph showing the number of viable cytoplasm (DiD+) cells that were not phagocytosed by macrophages (F4 / 80- and CD1 lb-), indicating that the cytoplasm escaped macrophage phagocytosis in the lungs. Mean ± SEM; n=3. Control cytoplasm labeled with DiD dye or engineered cytoplasm (mCD47 cytoplasm) was injected retroorbitally into the vascular system of mice. After 24 hours, tissue was collected and stained with two different pan-macrophage markers (F4 / 80 and CD1 lb). Figure 18D is a representative bar graph showing viable cytoplasm (DiD+) cells that were not phagocytosed by macrophages (F4 / 80- and CD1 lb-), indicating that the cytoplasm escaped macrophage phagocytosis in the liver. Mean ± SEM; n=3. Control cytoplasm labeled with DiD dye or engineered cytoplasm (mCD47 cytoplasm) was injected retroorbitally into the vascular system of mice. After 24 hours, tissue was collected and stained with two different pan-macrophage markers (F4 / 80 and CD1 lb). [Figure 19-1] Figure 19A is a schematic diagram of the IL-12 mRNA design. The Kozak sequence was appended before the start codon of the IL-12 mRNA coding region (CDS). The 5'UTR and 3'UTR of human betaglobin (HBB) mRNA were appended to the 5' and 3' ends of the IL-12 CDS, respectively. An artificial 5' cap was appended to the 5' end of the IL-12 mRNA to perform pseudouridine modification and enhance mRNA stability. Figure 19B is a representative line graph showing the time course of secreted IL-12 in conditioned medium of MSCs or MSC-derived cytoplasm transfected with IL-12 mRNA and then plated at 2.5 × 10⁴ cells / well in a 24-well plate. Cells were harvested at the point of CM, and the secreted IL-12 concentration was determined by ELISA. MSC only = CM from untransfected control. MSC IL-12 = CM from MSCs transfected with IL-12 mRNA. Cytoplasm IL-12 = Cytoplasm transfected with IL-12 mRNA. [Figure 19-2]Figure 19C shows immunoblots indicating the activation of phosphorylated / activated Stat4 (P-Stat4). Mouse splenocytes were treated for 30 minutes with the indicated CM, either in complete medium, purified IL-12 protein standard, or recovered from MSCs or cytoplasm processed as shown in Figure 19B. MSC complete medium = Mouse splenocytes treated in MSC complete culture medium. MSC IL-12 = Treated with CM from MSCs transfected with IL-12 mRNA. Cytoplasmic IL-12 = Treated with CM from cytoplasm transfected with IL-12 mRNA. Figure 19D is a representative scatter plot showing the concentration of secreted IL-12 cytokine per 1 mg of tumor protein. IL-12-processed or control cytoplasm processed as shown in Figure 19B was injected into established E0771 (mouse medullary mammary carcinoma) tumors growing in syngeneic C57BL / 6 mice. 48 hours after tumor injection, the animals were euthanized, tumor samples were collected, lysed, and analyzed by ELISA. PBS = Sample of a mouse injected with PBS. Cytoplasm = Sample of a mouse injected with an unmodified cytoplasm. Cytoplasmic IL-12 = Sample of a mouse injected with a cytoplasm modified to express the IL-12 cytokine. [Figure 20-1]Figure 20A is a scatter plot showing the multiplier of change in interferon-γ mRNA expression. MSC-derived cytoplasm engineered to express IL-12 or control cytoplasm without IL-12 was injected into established E0771 tumors growing in syngeneic C57BL / 6 mice. 48 hours after injection, the animals were euthanized, tumor samples were collected, lysed, and analyzed by real-time RT-PCR. PBS = Samples from mice injected with PBS. Cytoplasm = Samples from mice injected with unengineered cytoplasm. Cytoplasm IL-12 = Tumor samples from mice injected with cytoplasm engineered to express the IL-12 cytokine. Each dot represents a mouse tumor sample. Mean ± SEM; n=5. Figure 20B is a scatter plot showing the multiplier of change in PD-L1 mRNA expression. Cytoplasm derived from MSCs engineered to express IL-12 or control cytoplasm without IL-12 was injected into established E0771 tumors growing in syngeneic C57BL / 6 mice. 48 hours after injection, the animals were euthanized, tumor samples were collected, lysed, and analyzed by real-time RT-PCR. PBS = Samples from mice injected with PBS. Cytoplasm = Samples from mice injected with unengineered cytoplasm. Cytoplasm IL-12 = Tumor samples from mice injected with cytoplasm engineered to express the IL-12 cytokine. Each dot represents a mouse tumor sample. Mean ± SEM; n=5. [Figure 20-2]Figure 20C is a scatter plot showing the multiplier of change in CXCL9 mRNA expression. MSC-derived cytoplasm engineered to express IL-12 or control cytoplasm without IL-12 was injected into established E0771 tumors growing in syngeneic C57BL / 6 mice. 48 hours after injection, the animals were euthanized, tumor samples were collected, lysed, and analyzed by real-time RT-PCR. PBS = Samples from mice injected with PBS. Cytoplasm = Samples from mice injected with unengineered cytoplasm. Cytoplasm IL-12 = Tumor samples from mice injected with cytoplasm engineered to express the IL-12 cytokine. Each dot represents a mouse tumor sample. Mean ± SEM; n=5. Figure 20D is a bar graph showing the magnification of the change in E0771 subcutaneous tumor size in C57B1 / 6 mice that received intratumoral injection of 3 × 10⁶ IL-12-modified cytoplasm (cytoplasmic IL12 group) or PBS (PBS group) on days 11, 14, and 18 after tumor cell inoculation. Magnification of tumor size = tumor volume on day 20 / tumor volume on day 11. Mean ± SEM; n=5. [Figure 21-1] Figure 21A is an epifluorescence microscopy image taken 7 days after injection of Lifeact-RFP-expressing MSCs or cytoplasm infected with 0.05 MOI of oncolytic herpes simplex virus (oHSV-GFP) encoding GFP into subcutaneous U87 glioblastoma tumors in nude mice. Arrowheads represent RFP-positive cells (indicating MSC survival and proliferation within the tumor). Arrowheads represent GFP-positive tumor cells (indicating successful transfer of oHSV-GFP from MSCs or cytoplasm to tumor cells). Scale bar = 100 μm. [Figure 21-2]Figure 21B is a bar graph showing the percentage of GFP-positive tumor area for tumors treated as in Figure 21A, representing the portion of tumor cells infected with MSCs or cytoplasm carrying the oHSV-GFP virus. Figure 21C is a scatter plot showing the ratio of CD8+ effector T lymphocytes to total CD45+ (immune cells) present in tumors injected with engineered cytoplasm, as analyzed by flow cytometry. Established subcutaneous E0771 tumors in C57B1 / 6 mice were transfected with oHSV, and either IL-12 engineered cytoplasm or PBS alone (negative control) was injected into the tumors. [Figure 22] Figure 22A is a representative epifluorescence microscopy image showing RFP expression in nucleated MSC cells successfully fused with Cre-engineered cytoplasm. MSC-derived cytoplasm was genetically engineered to express Cre recombinase and then electrofused in a 3:1 ratio with hTERT-MSCs engineered to express Loxp-GFP-stop-Loxp-RFP (less than 500V for 100 μs in 3 pulses). Fluorescence images were taken after RFP sorting and Hoechst staining. Arrowheads indicate Hoechst-stained nuclei, and arrows indicate positive RFP fluorescence, showing successful Cre-inducible expression of RFP. Scale bar = 100 μm. Figure 22B is a bar graph showing the percentage of RFP+ (fused) cells out of total cells. Loxp MSCs only = Loxp-GFP-stop-Loxp-RFP-hTERT-MSCs in single-cell culture. Cre cytoplasm only = Single culture of cytoplasm manipulated to express Cre recombinase. Co-culture = 48 hours of co-culture of Loxp MSCs and Cre cytoplasm. Fusion = Electrofusion of Loxp MSCs and Cre cytoplasm. Mean ± SEM; n=4. [Figure 23] Figure 23 is a schematic diagram of a lentiviral vector engineered to express mCCR2 (SEQ ID NO: 20) in MSCs and cytoplasm. [Figure 24-1]Figure 24A is a representative scatter plot showing the number of DiD-labeled MSCs or cytoplasms detected in the lung. MSCs were cultured in suspension under standard adhesion conditions (2D) or the hanging-drop method (3D) to generate 3D cytoplasm. MSCs and cytoplasm were labeled with Vybrant® DiD dye and injected retroorbitally into the vascular system of C57BL / 6 mice. Tissue was collected after 24 hours, and the cell suspension was analyzed by flow cytometry. Mean ± SEM; n=2. Figure 24B is a representative scatter plot showing the number of DiD-labeled MSCs or cytoplasms detected in the liver. MSCs were cultured in suspension under standard adhesion conditions (2D) or the hanging-drop method (3D) to generate 3D cytoplasm. MSCs and cytoplasm were labeled with Vybrant® DiD dye and injected retroorbitally into the vascular system of C57BL / 6 mice. Tissue samples were collected 24 hours later, and the cell suspension was analyzed by flow cytometry. Mean ± SEM; n=2. [Figure 24-2] Figure 24C is a representative scatter plot showing the number of Vybrant® DiD-labeled MSCs or cytoplasms detected in the spleen. MSCs were cultured in suspension under standard adhesion conditions (2D) or the hanging-drop method (3D) to generate 3D cytoplasm. MSCs and cytoplasms were labeled with DiD dye and injected posteriorly into the vascular system of C57BL / 6 mice. Tissue was collected 24 hours later, and the cell suspension was analyzed by flow cytometry. Mean ± SEM; n=2. [Figure 25]Figure 25A is a representative line graph showing the viability of MSCs and MSC-derived cytoplasm immediately after recovery from cryogenic hibernation at 4 degrees Celsius for the indicated amount of time. Viability was assessed by automated cell counting using trypan blue exclusion and expressed as a ratio to the number of cells introduced. Figure 25B is a representative bar graph comparing transported MSCs and MSC-derived cytoplasm in a Boyden chamber assay immediately after recovery from cryogenic hibernation at 4 degrees Celsius for the indicated amount of time. Cells and cytoplasm were transported for 3 hours either without serum (negative control) or with 10% premium FBS (P-FBS) as a chemoattractant in the bottom chamber, and counts were normalized to the loading control. [Modes for carrying out the invention]

[0038] This disclosure relates to the therapeutic use of cells from which the nucleus has been removed (e.g., cytoplasm) as described herein. This demonstrates the function for the first time. In some embodiments, cells are treated with cytochalasin B. The cortical actin cytoskeleton can then be softened. The nuclei are then rapidly centrifuged under a Ficoll gradient. This allows for the physical extraction of cells from the cell body, thereby generating anucleated (enucleated) cytoplasm. Since the cellular and intact nucleated cells precipitate into different layers of the Ficol gradient, several implementations are possible. Morphologically, the cytoplasm is used for therapeutic purposes or for fusion with other cells (nucleated or enucleated). Therefore, it can be easily isolated and prepared. The enucleation process has been clinically expanded to thousands. It can process tens of thousands of cells. Proof-of-concept data shows that it can process cytoplasm and clinically relevant cells. As a homing vehicle for delivering the bazooka / payload, a healthy individual can be used for treatment (e.g., (Improvement of energy, recovery from exercise, or delivery of natural products), or various diseases (e.g.) This indicates that it can be used to treat any of the diseases described herein. For example, using cytoplasm to provide supplements, anti-aging factors, and preventive treatments to healthy individuals For example, if the delivered medication is not diagnosed as having a specific disorder for which it is effective. It can be delivered to an individual. The cytoplasm may have one or more of the following characteristics. Because it is possible to achieve this, it has significant therapeutic value: maintaining survival for up to 14 days, etc. They do not differentiate into any cell type, secrete bioactive proteins, and physically move / home. It is possible to manipulate it extensively ex vivo to perform specific therapeutic functions. , and fusion with the same or other cell types to achieve natural or manipulated desired cellular functions. It can transfer these molecules. Therefore, the cytoplasm is important for therapeutic biomolecules and gene editing. Factors, and chemotherapy drugs (e.g., doxorubicin), genes, viruses, bacteria, mRNA Disease targets including shRNA, siRNA, peptides, plasmids, and nanoparticles This novel cell vehicle for delivering ting cargo may have broad utility. In some embodiments, using the cytoplasm described herein may result in undesirable D Since NA is not expected to be transferred to the target, this will favorably enable the production of safe therapeutic drugs. In some embodiments, the present disclosure describes cytoplasmic cell death, and in some embodiments, positive Because it can occur within a specific timeframe, such as 3-4 days, it allows for more controllable treatment. In some embodiments, the cytoplasm described herein is a specific gene-edited cargo, disease Genes designed to deliver fighting cargo and health-promoting cargo to humans or animals. It can act as a manipulable cell-based carrier. Finally, large quantities of therapeutic materials for clinical application Because cell production is limited and can be expensive, many cells are produced, especially in the field of stem cells. The application of cell-based therapies is limited. Therefore, it may be robust and cost-effective. Therefore, using immortalized cells (using hTERT, viruses, and oncogenes) Increasing production capacity can be beneficial. However, immortalized cells can cause cancer. Yes, and therefore, it may be too dangerous for therapeutic application. Nucleated cells are administered or used Because it can be safely performed by enucleation beforehand, this disclosure relates to the large-scale production of cultures for therapeutic use. For the purpose of creation, nucleated cells (immortalized cells), cancer cells (e.g., any cancer cell), primary cells (e.g., incarcerated cells) You can use cells (primary origin) or cell lines.

[0039] This disclosure provides a safe and controllable cell-based therapeutic agent that maintains the nucleus throughout its lifespan in a target population. This invention provides a method for generating cells from any nucleated cell type that either retains or does not naturally undergo enucleation. In some embodiments, the present disclosure is derived from either normal or cancer cell lines (e.g.) For example, from any nucleated cells obtained therefrom, or, but not limited to, immune Commonly used therapeutic cells derived from (e.g., obtained from) a system (e.g., Na Natural killer (NK) cells, neutrophils, macrophages, lymphocytes, mast cells, basophils Spheres, eosinophils), stem cells (e.g., iPSCs (induced pluripotent stem cells), adult stem cells (e.g., interstitial cells) Leaf stem cells, embryonic stem cells, and fibroblasts extracted from the body This provides a method for removing the nucleus from any primary cell (also known as enucleation). This involves creating therapeutic cytoplasm that can survive for a limited period, for example, up to 3-4 days. Therefore, in some aspects of this disclosure, the following actions are possible: proliferation, differentiation, target Permanent engraftment, carcinogenesis, or transfer of nuclear-encoded DNA / genes into a target (For example, the introduction of dangerous DNA / genes encoded in the nucleus into a target) The novel use of cytoplasm as a safe therapeutic vehicle that cannot achieve multiple objectives. provide.

[0040] In the case of cell-based therapies, FDA approval is, in some cases, proof that the cells are stable. It is based on the idea that once it enters the target area, the cells may change, which can make it dangerous. This means that there are none. However, primary cells, irradiated cells, or "death switch" control cells. Current cell products, including those containing [specific components], have the potential to respond to or change in the in vivo microenvironment. It still has the potential to transcribe new genes. This gene mutation can still be retained, and this is not a controllable response in vivo. The copy prevents the ability to meet regulatory requirements. Conversely, a cytoplasm lacking a nucleus is generally... Even in significantly different in vivo microenvirons, there is no possibility of new gene transcription. Therefore, it is a more controlled and safer cell-based therapy.

[0041] To date, cell-based therapies generally involve normal or manipulated nucleated cells. It is being used. Some cell-based therapies induce cell proliferation and lethal DNA damage. To prevent damage, cells are irradiated before administration to the target. However, this method induces mutations. It generates a significant amount of reactive oxygen species that can irreversibly damage cellular proteins and DNA. This could release a large amount of damaged / mutated DNA into the target's body. Such products , if incorporated into other cells and / or trigger an undesirable anti-DNA immune response This can be dangerous. Furthermore, irradiated cells can transfer their mutated DNA to host cells through cell-cell fusion. And because genes can be introduced, it can be dangerous. Removing the entire nucleus from the cell This means that it can prevent any introduction of nuclear DNA into the target, thus limiting the cell's lifespan. It is a method with minimal risk and is extremely safe. Furthermore, it can be used with many stem cells, such as mesenchymal stem cells. Since mesenchymal stem cells (MSCs) are highly resistant to radiation-induced death, this method is used. It cannot be made safe. In other cases, therapeutic cells have a limited lifespan. It is being manipulated using a drug-induced suicide switch. However, the switch in vivo To activate Chi, it is necessary to administer potent and potentially harmful drugs that have undesirable side effects. This may be necessary. This method can induce cell suicide in cultured cells (for example, 95%). (Extremely), it is expected to be inefficient when translated clinically. Although not a binding mechanism, drug-induced suicide switches occur when all cells in the subject are affected by the drug. Since it is not subject to material-induced death, safety measures may be insufficient in clinical practice. It is thought that there are extensively manipulated cells or stem cells or cancer cells. In the case of cysts, drug-induced suicide switches are considered dangerous or insufficient for clinical practice. It is possible. Furthermore, the death of therapeutic cells results in a large amount of DNA (normal or genetically altered). It may release (of) which may be incorporated into host cells or cause dangerous systemic infections. It can induce an anti-DNA immune response. Cells may mutate and / or lose their suicide switch or If inactivated, these cells can become uncontrollably mutated. Furthermore, these cells are countered by In elephants, there is a possibility of fusion with host cells, and therefore DNA (e.g., mutant DNA) It is introduced. Not all host cells inherit the suicide gene, but chromosomal rearrangement During cell hybridization, it is possible to inherit a portion of the genes / DNA of the therapeutic cells. Because of this potential, such fused cells can be dangerous. Furthermore, for the same reason, Therapeutic cells containing a killer switch are used in vitro as cell fusion partners. It is not always ideal for this purpose. Another way to limit the lifespan of therapeutic cells is through heat-induced death. Therefore, this terminates beneficial biological functions (e.g., protein translation) in therapeutic use. This causes severe damage. Unlike cytoplasmic cells, nucleated cells, and the methods described above, Even in some cells that have been inactivated by this, the nucleus and genetic material are still retained. Naturally, DNA can be introduced into it. This includes chemotherapy drugs and mitomycin C, among others. A number of chemicals inhibit cell proliferation and / or cause cell death before therapeutic use. However, such drugs have significant off-target effects that severely damage cells. It may be present, and its high toxicity makes it undesirable for clinical use. Many antiproliferative drugs and kill Inducing drugs do not effectively inhibit 100% of cells due to resistance, and are different from the cytoplasm. Furthermore, the effects of many drugs are reversible. Therefore, this method is used for immortalized cells or cancer It is not suitable for inhibiting cell proliferation in vivo.

[0042] This disclosure does not limit the scope of DNA / genes (e.g., plasmids) RNA ( For example, mRNA, shRNA, siRNA, miRNA), proteins, peptides, small Molecular therapies (e.g., small molecule drugs), gene editing components, nanoparticles, and other therapeutic agents (For example, bacteria, bacterial spores, bacteriophages, bacterial components, viruses (for example, tumor lysis) A biomolecule with therapeutic function, containing (detoxifying viruses), exosomes, lipids, or ions. Method for generating therapeutic cytoplasm with spontaneous or induced expression and / or incorporation of offspring. To provide.

[0043] This disclosure does not limit itself to DNA / genes (e.g., plasmids), RNA. (e.g., mRNA, shRNA, siRNA, miRNA), proteins, peptides, Small molecule therapeutics (e.g., small molecule drugs), gene editing components, nanoparticles, and other Therapeutic agents (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses (e.g., tumors) Therapeutic cargoes containing cerebral lytic viruses, exosomes, lipids, or ions are targeted. This invention provides a method for using the cytoplasm as a vehicle for delivery.

[0044] This disclosure does not limit itself to DNA / genes (e.g., plasmids), RNA. (e.g., mRNA, shRNA, siRNA, miRNA), proteins, peptides, Small molecule therapeutics (e.g., small molecule drugs), gene editing components, nanoparticles, and other therapeutics Therapeutic agents (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses, exosolephates) Using the cytoplasm to create ions, molecules, compounds, complexes or living organisms (including lipids or other biological components). Methods for generating molecules (e.g., secreted, intracellular, inducible, or combinations thereof) It provides (what is possible).

[0045] This disclosure applies to any nucleated cell type (e.g., mammalian cells (e.g., human cells), or (Any mammalian cell as described herein), protozoan cell (e.g., amoeba cell), algae Cells of animals, plant cells, fungal cells, invertebrate cells, fish cells, amphibian cells, reptile cells, Large-scale therapeutic cytoplasm derived from (for example, obtained from) avian cells This provides a method for in vitro production. For example, cells can be naturally or genetically engineered. They may be immortalized and / or carcinogenically transformed by the process.

[0046] This disclosure does not limit itself to mitochondria, ribosomes, endosomes, and ribosomes. Sosomes, Golgi apparatus, DNA / genes (e.g., plasmids), RNA (e.g., mRNA) (shRNA, siRNA, miRNA), proteins (e.g., cytokines, growth factors) Children, and protein hormones), peptides, small molecule therapeutics (e.g., small molecule drugs), genes Editing components, nanoparticles, and other therapeutic agents (e.g., bacteria, bacterial spores, bacteria Phogmes, bacterial components, viruses (e.g., oncolytic viruses), exosomes, lipids, Organelles and biomolecules (secreted, intracellular, and native types) containing (or ions) To enhance and / or transfer (and inducible) other cells or cytoplasm (therapeutic or This uses therapeutic cytoplasm (natural or engineered) as a fusion partner to (natural) To provide a method.

[0047] This disclosure relates to the cryopreservation, cryopreservation, and storage of therapeutic cytoplasm in vitro. It provides a method for calling and recovering.

[0048] This disclosure relates to biosensors and systems for biological processes and health or disease conditions. This invention provides a method for using the cytoplasm as a signaling indicator for Gnar.

[0049] This disclosure also shows that in some embodiments, it can be used as a therapeutic agent, and / Or in a safe and controllable manner, specific disease-fighting cargo and health-promoting cargo A new drug that can be modified or genetically engineered to deliver to humans or animals. It is possible to generate standard anucleated cell-based products.

[0050] In the development of effective cell-based therapies, novel genetics in ex vivo are often required. Genetic manipulation and introduction of substances into the cell genome are necessary. However, this process is particularly When manipulated cells permanently engraft in the body or fuse with host cells, cancer and This could introduce dangerous mutations into the genome that could cause diseases that threaten the lives of other organisms. The indication is for the safe delivery of specific payloads, whether biological or synthetic. The entire nucleus from any nucleated cell type, for use as a whole therapeutic agent or vehicle. (That is, all the DNA encoded in the nucleus) can be removed. The cytoplasm Undesirable cases where a gene encoded in the nucleus or foreign or mutated DNA is introduced into the target. Since it does not cause a disease state and / or trigger an anti-DNA immune response, It may be safer than nuclear cells. This disclosure does not limit iPSCs (artificially pluripotent stem cells). Sex stem cells), any immortalized cells, stem cells, primary cells (e.g., host-derived cells), cell lines, Any immune cells, or any normal or manipulated nucleated cell type, including cancer cells. This can generate a new platform for safe annucleated cell therapy. It is noteworthy that the practical process was established in the literature more than 30 years ago. However, the therapeutic agent itself or any therapeutic cargo, whether natural or engineered, is not used. The use or development of cytoplasm as a vehicle for delivery to elephants has not been explicitly stated to date. not present.

[0051] A major problem with many existing cell-based therapies is that, after being delivered to the body, the cells become controlled. The problem is that they multiply uncontrollably, permanently engraft in the body, and potentially threaten life.

[0052] Furthermore, the lack of cell control after administration to the target makes it difficult to determine the correct dose of therapeutic cells and This may make it difficult to deliver the bioactive product (i.e., poor pharmacokinetics). In some embodiments, the cytoplasm has the same biological / therapeutic function as its nucleated equivalent. It can perform many of these tasks, but it may have a predetermined lifespan (e.g., 1 hour to 14 days). Furthermore, it neither proliferates nor permanently engrafts in the target. Therefore, the cytoplasmic body The pharmacokinetics of therapies are definable, and controllable and predictable responses in the subject are achieved. It is very safe.

[0053] In some embodiments of this disclosure, cytoplasm is deployed to a target beyond its predetermined lifespan. It can be administered (e.g., "dead" cytoplasm). For example, the process of administering dead cytoplasm It may have an immunostimulatory effect on the target.

[0054] Before delivery to the patient or subject, conventional cell-based therapeutics undergo the process of determining the desired cellular function and They are commonly modified or genetically altered ex vivo to gain therapeutic function. Furthermore, when these cells are introduced into a target, the new host environment is significantly reprogrammed. It can change to a negative value or otherwise neutralize them. The cytoplasm lacks a nucleus. Therefore, there is no new gene transcription, and in some embodiments, the cytoplasm regenerates This means that it cannot respond to programming and harmful external signals. Therefore When cytoplasm is introduced into a target, its differentiated phenotype and ex vivo It can maintain the therapeutic function that has been manipulated, resulting in a more controllable and predictable therapeutic vehicle. Similarly, normal donor cells that are directly enucleated will, when transplanted into the target, in The program / characteristics can be preserved in vivo. Overall, the cytoplasm is in It is possible to maintain phenotype and biological function both in vitro and in vivo. Therefore, it could become a more controllable and predictable therapeutic vehicle than conventional cell-based therapies. Such characteristics are found in newly induced (e.g., newly obtained) donor cells, or in specific types of donor cells. Many therapeutic applications rely on cells that have been manipulated ex vivo to perform specific therapeutic functions. This is extremely important.

[0055] Unlike nucleated cells, anucleated cytoplasm is used in some embodiments to treat cancer or other diseases. High-dose DNA damage / gene targeting for delivery to targets as a therapeutic agent for [condition] It can be loaded with drugs. This includes, but is not limited to, DNA damage chemotherapeutic drugs. DNA-integrated viruses, oncolytic viruses, and, but not limited to, cytotoxic viruses. cluster regularly interspaced short palindromic repeats (CRISPR) ), small clusters of Cas (CRISPR / Cas system), and plasmids This includes gene therapy application / delivery.

[0056] In some embodiments, the cytoplasm can also be loaded with any cargo. It does not, and can inherently produce a therapeutic effect when administered to the target. Several implementation forms In this state, the cytoplasm is not manipulated to produce one or more therapeutic agents, It may be. In some embodiments, the cytoplasm is loaded with cargo. Both can be therapeutic without being manipulated to produce one or more therapeutic agents. For example, an unprocessed cytoplasm itself may possess therapeutic properties when delivered to a patient or subject. In some embodiments, the unprocessed cytoplasm contains therapeutic DNA molecules and therapeutic RNA molecules. Children, therapeutic proteins, therapeutic peptides, small molecule therapeutics, and / or gene editing factors One or more of these can be generated. In some embodiments, untouched details Cholestaceans (e.g., those derived from oneself or the same source) have the following functions: therapeutic surface proteins, immunological Expression of pathogen-stimulating antigens or receptors, secretion of cytokines, hormones or proteins, Immunostimulatory activity through exosome release, membrane particle shedding, and death processes, or mitochondrial activity. Among the production of tunnel nanotubes into which biomolecules derived from ndria and other cells can be transferred It may have the ability to bring about one or more of the following: death The resulting cytoplasm can inherently produce therapeutic effects.

[0057] In some embodiments, the cytoplasm is applied to cells (e.g., xenocultured cells), or These can be cultured and their properties can be altered. For example, in some embodiments, Cytoplasm (e.g., unmanipulated cytoplasm or manipulated cytoplasm) is used in xenocultured cells. It can upregulate health-promoting factors, and in some cases, heterologous culture cells The cells can be returned to the object from which they were collected.

[0058] Unlike nucleated cells, the cytoplasm is susceptible to DNA damage-induced apoptosis. Therefore, apoptosis inducers and / or for treating cancer and other diseases Alternatively, it can be used in combination with DNA toxicity / targeting agents.

[0059] The cytoplasm is smaller than the nucleated equivalent, resulting in small openings in the vascular system and tissue parenchyma. It can move well through the section. Furthermore, the removal of large, high-density nuclei is a major physical This reduces the barrier, allowing cells to freely pass through small openings in blood vessels and tissue parenchyma. Therefore, the cytoplasm has improved in vivo distribution and reach of target tissues. It can be moved.

[0060] Unlike nucleated cells, the cytoplasm of the cytoplasm transforms into the same or different cell type of a similar or different origin. The fusion is not limited to cell surface proteins, signaling molecules, and secreted proteins. While maintaining desirable therapeutic characteristics, including protein, lipid, and epigenetic changes, This generates a unique cell hybrid without nuclear transfer.

[0061] Exosomes and small cell membrane vesicles derived from therapeutic cells can, in some cases, be used alone. Alternatively, it has been shown to have therapeutic effects as a delivery vessel, but cells It differs significantly from the cytoplasm and may be limited compared to the cytoplasm. Similarly, red blood cells (RBCs) RBCs (red blood cells) are hypothesized to be useful as a drug delivery system. Unlike the cytoplasm, it may have limitations. Exosomes, membrane vesicles, and RBCs Unlike other organelles, the cytoplasm is involved in many active biological processes and all cellular organelles. (For example, ER / Golgi, mitochondria, endosomes, lysosomes, cytoskeleton, etc.) ) can be a viable cell-like entity that can retain nucleated cells. Therefore, the cytoplasm is a nucleated cell. They function in ways that are important for biological functions, such as adhesion, tunnel nanotube formation, and activation. Electron-mediated diffusion (2D and 3D), transport, chemoattractant gradient sensitivity, mitochondrial translocation mRNA translation, protein synthesis, and secretion of exosomes and other bioactive molecules. These may indicate one or more of these functions, such as exosomes, small cell membrane vesicles, and This may not be shown by RBCs. Compared to RBCs derived from erythroblasts, cells The type of cell is not limited to iPSCs (induced pluripotent stem cells), any immortalized cells, Stem cells, primary cells (e.g., host-derived cells), cell lines, any immune cells, cancer cells, etc. They may originate from any type of nucleated cell, or any eukaryotic cell.

[0062] The limitations of developing cell-based therapeutics for clinical use are that a sufficient quantity of therapeutic cells is required. It may be impossible or inefficient to produce cells, especially stem cells, in this manufacturing process. To mitigate the "impairment," immortalized cells (hTERT, viruses, and oncogenes) are used. Therefore, methods have been considered to enhance cell regeneration capacity in a cost-effective manner. However, Immortalized cells have a higher risk of causing cancer, and therefore, they are a target for in vivo therapeutic purposes. It could be too dangerous for some, and is currently not approved by the Food and Drug Administration (FDA). No. Importantly, this disclosure does not apply to immortalized cells, cancer cells (e.g., any cancer cells), Primary (e.g., host-derived) cells, or cell lines for large-scale production of therapeutic cells are used. This is possible because such cells are denucleated before delivery and can be used as safe therapeutic agents. This is because... Furthermore, this disclosure relates to large-scale manufacturing and biotechnology for use in autotherapy or allotherapy. For banking purposes, more cells, cell lines, and / or immortalized cells are available for individual targeting. It can be generated from (see, for example, Figure 1). This makes it possible to produce commercially available products. This significantly improves the consistency and quality control of cell-based therapeutics that can be provided. ru.

[0063] Furthermore, since the cytoplasm is derived from cells (for example, obtained from cells), it cannot be artificially synthesized. It can be a superior therapeutic vehicle compared to nanoparticle and liposome formulations. Therefore, because it is a fully functional cell entity (excluding the nucleus), it reduces the toxicity of the target while Important physiological functions, cellular characteristics / organelles, and physiological ability to generate bioactive molecules To demonstrate strength.

[0064] In some embodiments of the methods, cytoplasm, and compositions described herein, nucleated cells (e.g., eukaryotic cells), mammalian cells (e.g., human cells, dog cells, cat cells) Horse cells, pig cells, primate cells, bovine cells, sheep cells, rodent cells (for example, ma (Uss cells, guinea pig cells, hamster cells, or mouse cells), immune cells, or Treat any nucleated cells described herein with cytochalasin B and the cortical actin cytoskeleton. The nucleus is then softened. Next, the nucleus is physically separated from the cell body by high-speed centrifugation using a Ficol gradient. Extract and produce anucleated cytoplasm. When used herein, the term “cytoplasm” also "Recombinant cytoplasm" is used interchangeably and consists of the internal mass of the cell and cellular organelles. This refers to anucleated cells obtained from the aforementioned nucleated cells (for example, any cells described herein). In some embodiments, the cytoplasm contains therapeutic DNA molecules, therapeutic RNA molecules, and therapeutic Expressing therapeutic proteins, therapeutic peptides, small molecule therapeutics, and / or gene editing factors Obtain. In some embodiments, the cytoplasm contains therapeutic DNA molecules, therapeutic RNA molecules, and therapeutic Therapeutic proteins, therapeutic peptides, small molecule therapeutics, and / or gene editing factors, nano It may contain particles or another therapeutic agent. In some embodiments, empty cytoplasm. A body (for example, a cytoplasmic body free of exogenous components) is used as a negative control.

[0065] In some embodiments, the cytoplasm is manipulated to, for example, chemokine receptors, adhesion molecules. This may improve the homing of the cytoplasm to the antigen or site within the target, or to achieve desired results. It is possible to express other markers that can stimulate and / or modulate the immune response. Yes, it is possible. For example, the cytoplasm can be manipulated to express anti-PD-L1 antibodies.

[0066] In some embodiments, nucleated cells are 3D (for example, in a suspension, as adherent cells) Can they be cultured (for example, as adherent cells in a semi-suspension or other non-adherent method)? Alternatively, clonal selection / enlargement can be performed before nucleation.

[0067] In some embodiments, the cytoplasm lasts for 1 hour to less than 14 days (for example, 1 hour to 1 hour) Less than, 1 hour to less than 6 hours, 6 hours to 12 hours, 12 hours to 1 day, 1 day, 2 days, 3 days, 4 Sunday, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 13th, 14th, 1st-14th, 1 ~12th, 1-10th, 1-9th, 1-8th, 1-7th, 1-6th, 1-5th, 1-4th , 1-3 days, 1-2 days, 2-14 days, 2-12 days, 2-10 days, 2-8 days, 2-7 days, 2 ~6 days, 2-5 days, 2-4 days, 2-3 days, 3-14 days, 3-12 days, 3-10 days, 3-8 days Sunday, 3-7 days, 3-6 days, 3-5 days, 3-4 days, 4-14 days, 4-12 days, 4-10 days, 4-8 days, 4-7 days, 4-6 days, 4-5 days, 4-7 days, 5-14 days, 5-12 days, 5-1 Day 0, Days 5-8, Days 5-7, Days 5-6, Days 6-14, Days 6-12, Days 6-10, Days 6-8 6-7 days, 7-14 days, 7-12 days, 7-10 days, 7-8 days, 8-14 days, 8-12 days , 8-10 days, 10-14 days, 10-12 days, 12-14 days, less than 14 days, less than 12 days, Less than 10 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days Having a predetermined lifespan of less than one day, less than 12 hours, or less than 6 hours. Several implementations Morphologically, the lifespan of a population of cytoplasmic bodies is determined by the lifespan of a portion of that population (e.g., at least of the population). 50%, at least 60%, at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 95%, or at least 98% Cell death can be evaluated by determining the average time at which cells are judged to have died. This can be determined by any method known in the art. In some embodiments For example, the viability of cytoplasm at one or more time points is a morphometric parameter or Whether functional parameters are intact (e.g., trypan blue dye exclusion, intact fine particles) Evaluation of vesicular membranes, evaluation of adhesion to plastics (e.g., in adherent cytoplasm), and cytoplasmic migration. By evaluating the results, such as by negative staining using apoptosis markers, the determination can be made. It can be evaluated. In some embodiments, the lifespan of the cytoplasm is the lifespan of the obtained fine particles. This may be related to the lifespan of cells. For example, in some embodiments, obtained from macrophages. The cytoplasm can survive for 12 to 24 hours.

[0068] In some embodiments, the cytoplasm is not a naturally occurring enucleated cell. In some embodiments, the cytoplasm is not obtained from cells that have undergone spontaneous enucleation. Morphologically, the cytoplasm is not that of a cell enucleated within the subject's body. In some embodiments, The cytoplasm cannot be obtained from cells that may have been enucleated within the subject's body. In the application method, cytoplasm cannot be obtained from erythroblasts. In some embodiments, cytoplasm This is obtained from cells that maintain their nucleus throughout their lifespan (for example, the enucleation method described herein). (If operations such as the above do not exist). In some embodiments, the cytoplasm is an anucleated cell (for example) For example, red blood cells (erythrocytes), platelets, lens cells, and It is not a cell found in the subject as a nucleated precursor immediately before it. In its natural state, the cytoplasm consists of the endoplasmic reticulum, Golgi apparatus, mitochondria, ribosomes, and proteasomes. It comprises one or more components selected from the group consisting of, or spliceosomes. In several embodiments, the cytoplasm has the following characteristics: adhesion, tunnel nanotube formation, and activation. Electron-mediated diffusion (2D and / or 3D), transport, chemoattractant gradient sensitivity, mitochondrial Rear import, mRNA translation, protein synthesis, and exosome and / or other biological processes. Characterized by the secretion of one or more microbially active molecules. In some embodiments, cells The organism is characterized by its ability to secrete proteins (e.g., using exosomes). In some embodiments, the cytoplasm is enucleated ex vivo. In this state, the cytoplasm is enucleated in vitro. In some embodiments, The cytoplasm is physically denucleated (for example, by centrifugation). In some embodiments The cytoplasm is an engineered enucleated cell. In some embodiments, the cytoplasm is red These are not blood cells. In some embodiments, the cytoplasm does not contain hemoglobin. In some embodiments, the cytoplasm does not have a biconcave shape.

[0069] In some embodiments, the cytoplasm is not obtained from erythroblasts. So, the cytoplasm is obtained from cells that have no potential to become red blood cells (erythrocytes). In some embodiments, the cytoplasm is obtained from lymphoid progenitor cells. The cytoplasm is obtained from lymphocytes. In some embodiments, the cytoplasm is mesenchymal It is obtained from stem cells (e.g., from bone marrow). In some embodiments, the cytoplasm is the endothelium. It is obtained from stem cells. In some embodiments, the cytoplasm is obtained from neural stem cells. In some embodiments, the cytoplasm is obtained from skin stem cells.

[0070] In some embodiments, the cytoplasm has a diameter of at least 1 μm. In the morphological configuration, the cytoplasm is larger than 1 μm in diameter. In some embodiments, the cytoplasm The body has a diameter of 1-100 μm (for example, 1-90 μm, 1-80 μm, 1-70 μm, 1 ~60μm, 1~50μm, 1~40μm, 1~30μm, 1~20μm, 1~10μm , 1~5μm, 5~90μm, 5~80μm, 5~70μm, 5~60μm, 5~50μm m, 5~40μm, 5~30μm, 5~20μm, 5~10μm, 10~90μm, 10 ~80μm, 10~70μm, 10~60μm, 10~50μm, 10~40μm, 10 ~30μm, 10~20μm, 10~15μm, 15~90μm, 15~80μm, 15 ~70μm, 15~60μm, 15~50μm, 15~40μm, 15~30μm, 15 (~20 μm). In some embodiments, the cytoplasm has a diameter of 10-30 μm. In some embodiments, the diameter of the cytoplasm is 5-25 μm (for example, 5-20 μm). , 5~15μm, 5~10μm, 10~25μm, 10~20μm, 10~15μm, 1 (5-25 μm, 15-20 μm, or 20-25 μm) In some embodiments The cytoplasm is not an exosome. It is not bound by any particular theory. However, in some cases, some cytoplasmic cells may be arranged to ensure good biological distribution, or to counter It is thought that being small enough to avoid being absorbed into an elephant's lungs could be advantageous. .

[0071] As used herein, the term “eukaryotic cell” means a cell that has a distinct membrane-bound nucleus. To taste. Such cells include, for example, mammals (e.g., rodents, non-human primates). (or humans), non-mammals (e.g., fish, birds, reptiles, or amphibians), invertebrates Examples include vertebrate, insect, fungal, or plant cells. In some embodiments, Eukaryotic cells include yeast cells, such as Saccharomyces cerevisiae. In some embodiments, eukaryotic cells are found in higher eukaryotes, such as mammals. These are bird, plant, or insect cells. In some embodiments, nucleated cells are primary cells. In some embodiments, nucleated cells are immune cells (e.g., lymphocytes (e.g., T cells, B cells, macrophages, natural killer cells, neutrophils, mast cells, halophils These include basal cells, dendritic cells, monocytes, bone marrow-derived suppressor cells, and eosinophils. Several implementations In this state, nucleated cells are phagocytes or leukocytes. In some embodiments, nucleated cells are , stem cells (for example, adult stem cells (for example, hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, mesenchymal stem cells) Cells, endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, testicular cells, embryonic stem cells These are induced pluripotent stem cells (iPS cells). In some embodiments, nucleated cells are progenitor cells. In some embodiments, the nucleated cells are derived from a cell line. In some embodiments, nucleated cells are suspension cells. In some embodiments, nucleated cells are cells that have been immortalized by the expression of oncogenes. In some embodiments, nucleated cells are subjected to human telomerase reverse transcriptase (hTERT). ) or immortalized by the expression of any oncogene. In some embodiments, Nuclear cells are patient- or subject-derived cells (e.g., autologous patient-derived cells or allogeneic patient-derived cells). ) In some embodiments, before the nucleated cells are enucleated using any of the enucleation techniques described herein and known in the art, the nucleated cells are transfected with a vector (e.g., a viral vector (e.g., a retroviral vector (e.g., a lentiviral vector), an adenovirus associated virus (AAV) vector, a vesicular stomatitis virus (VSV) vector), or a hybrid viral vector), or a plasmid). (VSV) vector), or a hybrid viral vector), or a plasmid). are transfected.

[0072] Methods of culturing cells (e.g., any of the cells described herein) are well-known in the art. Cells can be maintained in vitro under conditions favorable for inducing specific biological functions having therapeutic capabilities / benefits, including growth, proliferation, survival, differentiation, and / or, without limitation, three-dimensional culture, hypoxic environment, culture with a predetermined extracellular matrix component, treatment with a chemical substance, exposure to cytokines, growth factors, or any exogenous substance, natural or synthetic, that induces a specific desired cellular response. can be maintained in vitro under conditions favorable for inducing specific biological functions having therapeutic capabilities / benefits, including growth, proliferation, survival, differentiation, and / or, without limitation,

[0073] In some embodiments, enucleated (e.g., using any of the methods disclosed herein) cells of an already used or developing cell therapy can be used to form cytoplasts. Non-limiting examples of already used or developing cell therapies include chimeric antigen receptor-engineered T cells (CAR-T), treatment of cancer using NK or macrophages; treatment of inflammatory diseases including cancer, autoimmune diseases (e.g., Crohn's disease, rheumatoid arthritis, any type of arthritis, etc.), pancreatitis; applications in regenerative medicine, wound healing, bone or cartilage repair, etc.; Alzheimer's Treatment of diseases, cognitive disorders such as Parkinson's disease; treatment of graft-versus-host disease; gene therapy (for example) , sickle cell anemia, severe combined immunodeficiency (ADA-SCID / X-SCID), cystic line Muscular dystrophy, hemophilia, Duchenne muscular dystrophy, Huntington's disease, Parkinson's disease, high blood pressure High cholesterol, alpha-1 antitrypsin, chronic granulomatous disease, Fanconi anemia , or Gaucher disease); and treatment of infectious diseases, such as HIV, hepatitis, malaria, etc. These are some examples.

[0074] I do not wish to be bound by any particular theory, but rather by the theories that are already in use, For example, in cell therapies under development, enucleation is less affected by the target microenvironment of the cytoplasm. Therefore, it does not have a positive impact on the safety profile and / or therapeutic benefit of cell therapy. It is thought that this is possible. Furthermore, in some embodiments, such cytoplasm is It can be operated using any of the methods described in the details. For example, several implementations Morphologically, such cytoplasm contains therapeutic DNA molecules, therapeutic RNA molecules, and therapeutic proteins. To express proteins, therapeutic peptides, small molecule therapeutics, and / or gene editing factors. It can be manipulated. In some embodiments, the cytoplasm is used to control therapeutic DNA molecules, therapeutic RNA molecules for therapeutic use, therapeutic proteins, therapeutic peptides, small molecule therapeutics, and / or genetics It may contain sub-editing factors, nanoparticles, or other therapeutic agents. Several embodiments So, what are the functions of such cytoplasmic cells? For example, chemokine receptors, adhesion molecules, antigens, or... To improve cytoplasmic homing to sites within the elephant, or to stimulate a desired immune response and / or Alternatively, it can be manipulated to express other markers that can be modulated. For example, the cytoplasm can be manipulated to express an anti-PD-L1 antibody.

[0075] In some embodiments of any of the compositions and methods provided herein, the cytoplasm The body is cooled or frozen for later use. Various methods of preserving cells are limited. Although not a product, serum (e.g., fetal bovine serum) and dimethyl methyl Use of rufoxide (DMSO) or dormancy medium for storage at 4°C (freeze-hibernation). Any of the compositions and methods provided herein are known in the art, including their use. In some embodiments, the cytoplasm is thawed before use.

[0076] Biomolecules (e.g., RNA molecules (e.g., mRNA, miRNA, siRNA, shR) NA (IncRNA), DNA molecules (e.g., plasmids), proteins, gene editing factors (For example, CRISPR / Cas9 gene editing factors, peptides, plasmids) into cells Used for introduction into a cell (for example, a cytoplasm derived from any cell described herein). Various methods are known in the art that can be used to introduce biomolecules into the cytoplasm. Non-limiting examples of methods that can be used include electroporation, my Chloroinjection, lipofection, transfection, calcium phosphate Transfection, dendrimer-based transfection, cationic polymers Transfection, cell squeezing, sonoporation, phototransfection Impalection, hydrodynamic delivery, magnetofection Examples include nanoparticle transfection.

[0077] In some embodiments of any of the methods and compositions described herein, the introduction further comprises expressing a somatic molecule within the endosome. A variety of expression vectors are known in the art and can be used herein. Non-limiting examples of expression vectors are provided herein. In some embodiments, the expression vector is the vector shown in any one of FIGS. 16A, 17A, 18A or 23. Various gene editing factors are known in the art. Non-limiting examples of gene editing factors include CRISPR / Cas9 gene editing, transcription activator-like effector nuclease (TALEN), and zinc finger nuclease.

[0078] In some embodiments of any of the compositions and methods provided herein, a therapeutic agent, virus, antibody, drug, or nanoparticle is introduced into the endosome. In some embodiments, therapeutic DNA, therapeutic RNA, therapeutic protein (e.g., enzyme, antibody, antigen, toxin, cytokine, protein hormone, growth factor, cell surface receptor, or vaccine, or any therapeutic protein available or under development), therapeutic peptide (e.g., peptide hormone or antigen, or any therapeutic peptide available or under development), small molecule therapeutic agent (e.g., steroid, polyketide, alkaloid, toxin, antibiotic, antiviral agent, analgesic, anticoagulant, antidepressant, anticancer agent, antiepileptic drug, antipsychotic drug, sedative, colchicine, taxol, mitomycin, emtansine, or any small molecule therapeutic agent available or under development), therapeutic gene editing factor, therapeutic <000097 {0}> ​​​​​​​​​​​​​​​​Nanoparticles, or other therapeutic agents (e.g., bacteria, bacterial spores, bacteriophages, bacterial components) , viruses (e.g., oncolytic viruses), exosomes, lipids, or ions in cells It is introduced into the substance.

[0079] In some embodiments, the cytoplasm is treated with exosomes (e.g., stimulated or loaded). ) can be done. In some embodiments, processing with exosomes is used to produce raw Somatic molecules, therapeutic drugs, therapeutic peptides, small molecule therapeutic drugs, therapeutic gene editing factors, therapeutic nanoparticles Children, or other therapeutic agents (e.g., bacteria, bacterial spores, bacteriophages, bacterial components, viruses) Rus (e.g., oncolytic viruses), exosomes, lipids, or ions are introduced into the cytoplasm. It can be introduced. In some embodiments, processing with exosomes is used to further refine It can alter the behavior of the cytoplasm, signal transduction, secretion factors, or other properties.

[0080] This method is used to identify diseases in the target population (e.g., cancer / neoplasms, infectious diseases, inflammatory conditions, neurological diseases). For example, neurodegenerative diseases, degenerative diseases, autoimmune diseases, cardiovascular diseases, ischemic diseases, and hereditary diseases. This includes genetic disorders, developmental disorders, ophthalmic diseases, bone diseases, metabolic diseases, poisoning, idiopathic conditions, and This includes the use of cytoplasm to treat two or more of those conditions.

[0081] Non-specific examples of cancer include acute lymphoblastic leukemia (ALL) and acute myeloid leukemia. (AML), adolescent cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, dysmorphic teratoma Rhabdomorphic tumors, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumor, breast cancer Cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumors, unknown primary cancers, cardiac tumors, Cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CLL) ML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphocytes Tumors, bile duct cancer, non-invasive ductal carcinoma, embryonic tumors, endometrial cancer, ependymoma, esophageal cancer, sensory neuroblastoma Tumors, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic cholangiocarcinoma, eye cancer, egg Tubal cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor Tumors (GIST), germ cell tumors, gestational trophoblastic disease, gliomas, glioblastomas, hirsutomas Cerebrospinal fluid, hair cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's disease, lymphoma Tumors, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, Kaposi's sarcoma, kidney cancer Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cancer, liver cancer, lung cancer Cancer, lymphoma, macroglobulinemia, malignant fibrous histiocytoma of bone, bone cancer, melanoma, Mel Kell cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma, midline carcinoma, oral cancer, multiple endocrine neoplasms Syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, Myeloid leukemia, multiple myeloma, myeloproliferative neoplasms, nasal cavity and sinus cancer, Nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer Lip cancer, oral and pharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, sinuses Cavity and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary cancer, phenotype Cellular tumors, pleuroblastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer , prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, solid tumors, squamous cell carcinoma Skin cancer, cervical squamous cell carcinoma, gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma and Thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, unknown primary cancer, urethral cancer, uterine These include uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor. In terms of disease type, cancer can be primary (e.g., primary tumor) or metastatic (e.g., metastatic tumor). could be.

[0082] Infectious diseases can be broadly categorized into viral infections, bacterial infections, fungal infections, and parasitic infections. Infectious diseases and protozoal infections are examples. An unspecified example of an infectious disease is Acinetobacter Acinetobacter infection, actinomycosis, African sleeping sickness (African trypanosomiasis) ), AIDS (Acquired Immunodeficiency Syndrome), Amebiasis, Anaplasmosis, Schistosomiasis, Ani Sakiasis, anthrax, hemolytic alkanobacterium (Arcanobacterium haemolyticum) infection Argentine hemorrhagic fever, roundworm infection, aspergillosis, astrovirus infection, babesiosis Bacillus cereus infection, bacterial pneumonia, bacterial vaginosis, Bacteroides (Bacteroides) infections, balanthidiasis, bartonellosis, Bayliscaris (Baylisasc) aris infection, BK virus infection, Black piedra, blastosis Blastocystosis, blastomycosis, originating in Bolivia Blood fever, botulism (and infant botulism), Brazilian hemorrhagic fever, brucellosis, glandular fever Plague, Burkholderia infection, Buruli ulcer, calicivirus infection Norovirus and sapovirus infections, Campylobacteriosis, Candidiasis (Moniliosis) Thrush, trichozoa, carrion's disease, cat scratch disease, cellulitis, Chagas disease (American) Catrypanosomiasis, chancroid, chickenpox, chikungunya fever, chlamydia, pneumoniae chlamydia (Chlamydophila pneumoniae) infection (Taiwan Acute Respiratory Factor or TWAR), cholera, Black cytoblastosis, chytridiomycosis, liver fluke infection, Clostridium difficile Difficile) Colitis, coccidioidomycosis, Colorado tick fever (CTF), common cold (acute viral infection) Rustic nasopharyngitis (acute rhinitis), Creutzfeldt-Jakob disease (CJD), Crimean rhinitis Ngo hemorrhagic fever (CCHF), cryptococcosis, cryptosporidiosis, cutaneous larval migration Clemens disease (CLM), cyclosporiasis, cysticercosis, cytomegalovirus infection, dengue fever, Desmodesmus infection, dinuclear amebiasis, diphtheria, phyllobothrium tapeworm infection, mediocresis Natulosis, Ebola hemorrhagic fever, echinococcosis, ehrlichiosis, pinworm infection, enteric worm Bacterial (Enterococcus) infections, enterovirus infections, epidemic typhus, erythema infectiosum ( (5th disease), sudden rash (6th disease), hepatosis, hypertrophic paragonimiasis, fatal familial insomnia (FFI) , filariasis, food poisoning caused by Clostridium perfringens, free-living (F) (ree-living) Amebic infection, Fusobacterium infection, gas gangrene (Clostridium erythropoiesis) Diamyloid myonecrosis, geotrichumosis, Gerstmann-Streusler-Scheinker disease GSS syndrome, Giardiasis, Glanders, Gnathostoma, Gonorrhea, Granuloma venereum (Donovan) (Symptoms), Group A Streptococcal infection, Group B Streptococcal infection, Haemophilus infection Hand, foot, and mouth disease (HFMD), Hantavirus pulmonary syndrome (HPS), Heart-related illnesses Virus disease, Helicobacter pylori infection, hemolytic uremic disease syndrome (HUS), hemorrhagic fever with renal syndrome (HFRS), hepatitis A, hepatitis B, hepatitis C, hepatitis D Hepatitis, Hepatitis E, Herpes simplex, Histoplasmosis, Hookworm infection, Human bocavirus infection, Human ehrlichiosis, human granulocytic anaplasmosis (H GA), human immunodeficiency virus (HIV) infection, human metapneumovirus infection, hi Tomonocytic ehrlichiosis, human papillomavirus (HPV) infection, human parainfluenza Enzavirus infection, membranoid tapeworm infection, Epstein-Barr virus infectious mononucleosis (Mo No), influenza (flu), isosporiasis, Kawasaki disease, keratitis, Kingella keratitis Kingella kingae infection, Kuru disease, Lassa fever, Legionnaires' disease Veterans disease (Pontiac fever), leishmaniasis, leprosy, leptospirosis, Listeriosis, Lyme disease (Lyme borreliosis), lymphangiofilariasis (elephantiasis), phosphorus Pachycytic choriomeningitis, malaria, Marburg hemorrhagic fever (MHF), measles, Middle East respiratory syndrome ( MERS, meridian (Whitmore's disease), meningitis, meningococcal disease, metagonimus, Microsporiasis, molluscum contagiosum (MC), monkeypox, mumps, typhus (localized typhus), malignant malaria Icoplasma pneumonia, Mycoplasma genitalium infection, Mycotoma (uniform), myiasis, neonatal conjunctivitis (neonatal ophthalmitis), norovirus (children and Infants, (new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), nocardi Anematocystitis, onchocerciasis (river blindness), opistolicis, paracoccidioidomycosis (southern American blastomycosis, lung fluke infection, pasteurellosis, head lice parasitic infection (head Lice, body louse parasitism (human lice), pubic louse parasitism (genital lice, pubic lice) Pelvic inflammatory disease (PID), pertussis (whooping cough), plague, pneumococcal infection, pneumococcal disease Cystic pneumonia (PCP), pneumonia, polio, Prevotella infection, primary Amebic meningoencephalitis (PAM), progressive multifocal leukoencephalopathy, psittacosis, Q fever, rabies, relapse Fever, respiratory syncytial virus infection, linosporidia, rhinovirus infection, ricketts Infections, rickettsial pox, Rift Valley fever (RVF), Rocky Mountain spotted fever (RMSF), Rotavirus infection, rubella, salmonellosis, SARS (Severe Acute Respiratory Syndrome), scabies, Scarlet fever, schistosomiasis, sepsis, dysentery (bacterial dysentery), shingles (herpes zoster), smallpox (Smallpox), sporotrichosis, staphylococcal food poisoning, staphylococcal infection, strongyloidiasis, subacute Sclerosing panencephalitis, syphilis, tapeworm infection, tetanus (trismus), tinea folliculitis (barber rash), tinea capitis ( Tinea corporis (ringworm of the body), tinea cruris (ringworm of the genitals), tinea manuum (ringworm of the hands), tinea pedis , tinea pedis (athlete's foot), onychomycosis (nail fungus), tinea versicolor (black catfish), toxocariasis (ocular larval transmission) Toxocariasis (OLM), toxocariasis (visceral larval migrans (VLM)), toxoplasmosis, Lacoma, trichinellosis, trichomoniasis, whipworm infection, tuberculosis, tularemia, typhoid fever , typhus, Ureaplasma urealyticum infection, Valley fever, Venezuelan encephalitis, Venezuelan hemorrhagic fever, Vibrio vulnificus (Vibrio vulnificus) Vibrio ulnificus infection, Vibrio parahaemolyticus enteritis, Viral pneumonia, West Nile fever, white sand trichomes (ringworm), Yersinia pseudotuberculosis Yersinia pseudotuberculosis infection, Yersinia disease, yellow fever, Zika fever and contact Mycosis is one example.

[0083] Non-specific examples of neurological diseases include amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Bell's palsy, cerebral aneurysm, brain injury, brain tumor, cerebral palsy, chronic fatigue syndrome, concussion, dementia, Epilepsy, Guillain-Barré syndrome, headache, Huntington's disease, migraine, multiple sclerosis, muscular dystrophy Lophii, neuralgia, neuropathy, neuromuscular and related disorders, Parkinson's disease, mental state (For example, depression, obsessive-compulsive disorder), scoliosis, seizures, spinal cord injury, spinal malformations, spinal cord disorders ( Examples include subacute complex degeneration, spinal tumors, stroke, and dizziness.

[0084] Non-specific examples of autoimmune diseases include achalasia, Addison's disease, and adult-onset Still's disease. Agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, auto Immunoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis , autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, axon and Neuropathies (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, water Bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory bowel disease Myelin polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), churg- Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), pemphigoid scarring, Cancer syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome Crohn's disease, herpetic dermatitis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetes (examples) For example, type 1 diabetes, type 2 diabetes, gestational diabetes, lupus discoid, Dresser syndrome. Endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed erythema Oglobulinemia, Evans syndrome, fibromyalgia, fibrotic alveolitis, giant cell arteritis (lateral) Cranial arteritis, giant cell myocarditis, glomerulonephritis, Goodpasture syndrome, and polyangiitis. Granulomatous thyroiditis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch's disease. Schönlein's purpura (HSP), herpes zoster of pregnancy or pemphigoid of pregnancy (PG), suppuration Hidradenitis genitalia (HS) (reverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis ( IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, rheumatoid arthritis Burt-Eaton syndrome, leukocytosis-destroying vasculitis, lichen planus, lichen sclerosing, woody conjunctivitis, Linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiopathy Inflammation (MPA), mixed connective tissue disease (MCTD), Mohren's ulcer, Mucha-Habermann disease Multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis Myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular scarring, etc. Pemphigus, optic neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (P CD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsplanitis (Peripheral uveitis), Personage-Turner syndrome, Pemphigus, Peripheral neuropathy, Static Peripulsive encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome Group I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, Post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone-induced dermatitis Psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, Reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, lower limb immobility Rheumatic syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, schemin Sjögren's syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, generalized rigidity Syndrome (SPS), subacute bacterial endocarditis (SBE), Suzak syndrome, sympathetic ophthalmitis (S O), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Toro Hunt syndrome (THS), transverse myelitis, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegena - Granulomatous disease (or granulomatous disease with polyangiitis (GPA)) is one example.

[0085] Non-specific examples of cardiovascular disease include acute myocardial infarction, heart failure, refractory angina, and coronary artery disease. Diseases, rheumatic heart disease, congenital heart disease, stroke, aortic aneurysm and / or dissection, peripheral artery Diseases, deep vein thrombosis, pulmonary embolism, cardiac tumors, cerebrovascular tumors, cardiomyopathy, heart valve disease, and Pericardial disease is one example.

[0086] Non-specific examples of ophthalmic diseases include glaucoma, cataracts, macular degeneration, diabetic retinopathy, and strabismus. Retinal detachment, uveitis, amblyopia, dry eye syndrome, keratitis, macular edema, corneal ulcer, optic nerve Symptoms, cytomegalovirus retinitis, corneal dystrophy, anterior chamber hemorrhage, trachoma, central serous retinopathy, retinopathy of prematurity, endophthalmitis, Leber congenital amaurosis, central retinal artery occlusion, eyelids Trichiasis, papilledema, Graves' ophthalmopathy, uveal melanoma, retinal vein branch occlusion, choroidal hemorrhage Examples include macular degeneration and macular degeneration.

[0087] Non-specific examples of bone diseases include osteochondrodysplasia, chondrodysplasia, and hypophosphatasia. , achondroplasia, thanatrophoric dysplasia, osteomalacia, rickets, bone loss Dermatitis, osteoporosis, Paget's disease, osteomyelitis, osteolysis, Hadzcz-Cheney syndrome, hypertrophic lung Osteoarthropathy, nonossifying fibroma, pseudoarthrosis, fibrous dysplasia, hyperosteopathy, osteosclerosis, and paraplegia Nodystosis is one example.

[0088] Non-specific examples of metabolic diseases include cystinuria, Fabry disease, and galactosemia. Gaucher disease (Type 1), Hartnapp disease, homocystinuria, Hunter syndrome, Hurler's disease Syndromes, Lesch-Nyhan syndrome, maple syrup urine disease, Maloto-Lamy syndrome, Morquio syndrome, Niemann-Pick disease (type A), phenylketonuria, Pompe disease, porphyria Phosphate syndrome, Schayet syndrome, Tay-Sachs disease, tyrosinemia (hepatorheological), von Gierke Examples include glycogen storage deficiency type 1A and Wilson's disease.

[0089] In some embodiments, the subject is in need of cytoplasmic therapy. It has been determined or is suspected to require it. In some embodiments, cancer Examples include acute myeloid leukemia, bladder cancer, breast cancer, kidney cancer, melanoma, small cell lung cancer, It could be non-small cell lung cancer, pancreatic cancer, or prostate cancer.

[0090] In some embodiments, the cytoplasm is a disease or condition (e.g., cancer or neoplasm). Infections, inflammatory conditions, neurological disorders (e.g., neurodegenerative diseases), degenerative diseases, autoimmune diseases, cardiovascular diseases Tube diseases, ischemic diseases, hereditary or genetic disorders, developmental disorders, ophthalmic diseases, bone diseases, metabolic diseases It can be used to diagnose diseases, poisonings, idiopathic conditions, or two or more of these. Therefore, this specification does not indicate specific health, disease, condition, or toxin levels of the subject. The cytoplasm described herein (for example, an unmanipulated cytoplasm, or a genetically modified cytoplasm) can be manipulated. Either created, or a bioreporter molecule or inducible bioreporter molecule is exogenously A method for diagnosing a subject, which includes the step of administering to one of the cytoplasms loaded into the device. A law or method for determining the presence or absence of disease or condition in a subject is provided. In this embodiment, molecules expressed or secreted by the cytoplasm and / or cytoplasm Molecules contained in the cytoplasm, or other similar molecules, can function as bioreporters. Bioreporters can be used in subjects or ex vivo. In some embodiments, a sample (e.g., blood, urine, stool, or tissue (e.g., biopsy)) It can be obtained from the subject. In some embodiments, the cytoplasm is, for example, measurable Colorimetric molecules, fluorescent molecules, luminescent molecules, chemiluminescent molecules, or electrochemical molecules that report clinical signals. It may express or contain molecules. The signal may be a chemical, physical substance, or biological substance. Is it possible to achieve a proportional relationship with the concentration of somatic molecules (e.g., growth factors, insulin, cancer antigens, immune factors)? Alternatively, gene transcription activity or protein translation activity in the target or sample derived from the target. It can be proportional to sex.

[0091] As used herein, the term "subject" means any living organism. For example, subject This includes mammals, amphibians, fish, reptiles, invertebrates, birds, plants, archaea, fungi, or It may be a bacterium. In some embodiments, the subject is a mammal. The subjects are rodents (e.g., mice, rats, hamsters, guinea pigs) and canids. (For example, dogs), felines (for example, cats), equids (for example, horses), sheep, cows, pigs, Non-human primates, such as primates (e.g., monkeys), great apes (e.g., gorillas, chimpanzees, It may be an orangutan, gibbon, or human. Any of the methods described herein. In some embodiments, the subjects are 0 to 120 years old (e.g., the first month after birth) (e.g., newborns). Newborns, 1 month to 2 years old (e.g., infants), 2 to 12 years old (e.g., children), 12 to 16 years old (For example, young people), 1-120 years old, 1-115 years old, 1-110 years old, 1-105 years old, 1-10 0 years old, 1-95 years old, 1-90 years old, 1-85 years old, 1-80 years old, 1-75 years old, 1-70 years old, 1 ~65 years old, 1~60 years old, 1~50 years old, 1~40 years old, 1~30 years old, 1~25 years old, 1~20 years old , 1-15 years old, 1-10 years old, 5-120 years old, 5-110 years old, 5-100 years old, 5-90 years old, 5-60 years old, 5-50 years old, 5-40 years old, 5-30 years old, 5-20 years old, 5-10 years old, 10-1 20 years old, 10-110 years old, 10-100 years old, 10-90 years old, 10-80 years old, 10-60 years old 10-10 years old, 10-50 years old, 10-40 years old, 10-30 years old, 10-20 years old, 20-1 20 years old, 20-110 years old, 20-100 years old, 20-90 years old, 20-70 years old, 20-60 years old 20-50 years old, 20-40 years old, 20-30 years old, 30-120 years old, 30-110 years old, 30 ~100 years old, 30-90 years old, 30-70 years old, 30-60 years old, 30-50 years old, 40-120 years old Ages 40-110, 40-100, 40-90, 40-80, 40-60, 4 0-50 years old, 50-120 years old, 50-110 years old, 50-100 years old, 50-90 years old, 50- 80 years old, 50-70 years old, 50-60 years old, 60-120 years old, 60-110 years old, 60-100 years old Ages 60-90, 60-80, 60-70, 70-120, 70-110, 7 0-100 years old, 70-90 years old, 70-80 years old, 80-120 years old, 80-110 years old, 80- 100 years old, 80-90 years old, 90-120 years old, 90-110 years old, 90-100 years old, 100- 120 years of age (or 110-120 years of age). Any of the methods described herein. In that embodiment, the subject has not yet been born and is, for example, in the womb. In some embodiments of the method, the subjects are at least 1 month old (e.g., young (at least 2 years old, at least 12 years old, at least 16 years old, or at least 18 years old) Using any of the methods described herein, a subject, for example, a diseased subject (that is, (For example, subjects with a disease, or subjects diagnosed with a disease), or asymptomatic subjects. In other words, subjects who demonstrate clinical health or who have not been diagnosed with a disease. It can treat the disease. When used herein, the treatment involves the risk of the disease. The aim is to reduce or prevent (or reduce the risk of) the incidence of signs or symptoms of disease in elephants. The "preventive treatment" that tastes good, and the reduction of signs or symptoms of the disease in subjects diagnosed with the disease, This includes "therapeutic treatment," which means reducing disease progression, disease severity, and recurrence. When used in a specification, the term “treat” means treating at least one clinical parameter of a disease. To improve the body and / or have beneficial effects (e.g., anti-aging, anti-scarring, wound healing, It means providing antidepressant, anti-inflammatory, and weight-loss benefits.

[0092] As used herein, “disease,” “disorder,” and “condition” refer to differences in the subject matter. This refers to any deviation from the normal or subjective state of health. It also refers to disease and / or condition. Non-specific examples include cancer or neoplasms, infections, inflammatory conditions, and neurological disorders (e.g., nerves). Degenerative diseases, autoimmune diseases, cardiovascular diseases, ischemic diseases, hereditary or genetic disorders These include harm, developmental disorders, ophthalmic disorders, bone disorders, metabolic disorders, poisoning, or idiopathic conditions.

[0093] In some embodiments of the methods provided herein, the composition, during the period, At least once (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 9 Administered 0 to 100 times (for example, daily, every other day, twice a week, once a week, weekly, three times a month) Twice a month, once a month, every two months, every three months, every four months, every five months, every six months (Every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or once a year). Also, monthly treatment, for example, at least once a month for at least one month (for example, at least Both 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months Administering the medication for more than a month, for example, 12 months or more, and annual treatment (for example, every year or so) Administration (once a year) during the above period is possible. Administration may be via any route known in the art. For example, subcutaneous, intravenous, intraarterial, intraocular, oral, intramuscular, intranasal (e.g., inhalation), and intraperitoneal. , local, mucosal, epidural, sublingual, supracutaneous, extra-amniotic, intra-articular, intradermal, intraosseous, intrathecal, child Intrauterine, intravaginal, intravesical, intravitreal, perivascular, and / or rectal administration, or known administration This can be done through any combination of the given methods.

[0094] In some embodiments, the cytoplasmic cell death process may have a therapeutic effect on the target. For example, in some embodiments, the cytoplasmic cell death process may be immunostimulatory. Therefore, this specification provides a method of administration targeting the cytoplasm, which is the death of the cytoplasm. A method is provided in which the treatment has a therapeutic effect on the target. In some embodiments, the target The cytoplasm administered to the target is dead. In some embodiments, the cells administered to the target If administered, the effect lasts for less than 5 days (for example, less than 4 days, less than 3 days, less than 2 days, or 36 hours). Less than, less than 1 day, less than 18 hours, less than 12 hours, less than 6 hours, less than 2 hours, or 1 hour It has a remaining lifespan of less than ).

[0095] In some embodiments, cells can be removed from the subject and enucleated. Morphologically, cells, before enucleation, (for example, therapeutic DNA molecules, therapeutic RNA molecules, Therapeutic proteins, therapeutic peptides, small molecule therapeutics, therapeutic gene editing factors, therapeutic nano Particles and / or other therapeutic agents are manipulated to be produced or contain them. In this embodiment, cells derived from the target are enucleated, and then (for example, therapeutic DNA molecules, therapeutic RNA molecules for therapeutic use, therapeutic proteins, therapeutic peptides, small molecule therapeutic drugs, therapeutic gene editing agents (Manipulated to produce or contain children, therapeutic nanoparticles, and / or other therapeutic agents) In some embodiments, the cytoplasm (whether or not they have been manipulated) The cells are administered to the target from which they were extracted.

[0096] In some embodiments, the culture medium in which the cytoplasm is cultured and / or stored ("conditioned medium") ) may have therapeutic benefits. In some embodiments, the cytoplasm (for example, after enucleation) ) Cells co-cultured and / or stored in culture media ("conditioned media") have therapeutic benefits. Obtain. In some embodiments, the cytoplasm fused with the cell is cultured and / or retained with the cell. The culture medium ("conditioned medium") may have therapeutic benefits.

[0097] Therefore, in this specification, the procedure includes the step of administering a conditioned medium to the subject. A method of treatment, prevention, or preventive treatment or health promotion is provided. Although not bound by theory, in some embodiments, the therapeutic benefits of the culture medium This is because exosomes (for example, containing therapeutic proteins) secreted by the cytoplasm are cultured. This is thought to be due to its presence underground.

[0098] In some embodiments of any of the methods provided herein, the composition is one or This involves multiple additional therapies (e.g., any medications (e.g., antibiotics, antivirals, anti-inflammatory drugs)). ) or chemotherapy (for example, chemotherapeutic agents (for example, doxorubicin, paclitaxel, cyanoacrylate) Clophosphamide)) or any of the small molecule therapeutic agents described herein), cell-based Therapies, radiotherapy, immunotherapy, small molecules, inhibitory nucleic acids (e.g., antisense RNA, antisense RNA) (Cysens DNA, miRNA, siRNA, IncRNA), exosome-based therapies It is administered in conjunction with gene therapy or surgery.

[0099] In some embodiments provided herein, the composition comprises one or more additional therapeutic agents. Laws (for example, any drugs (for example, antibiotics, antivirals) or chemotherapy (for example, Chemotherapy agents (e.g., doxorubicin, paclitaxel, cyclophosphamide), cells Base therapies, radiotherapy, immunotherapy, small molecules, inhibitory nucleic acids (e.g., antisense RN) A, antisense DNA, miRNA, siRNA, IncRNA (or surgery) It is included in.

[0100] Furthermore, in this specification, cytoplasm (for example, fine cells obtained from any of the cells described herein) Compositions (e.g., pharmaceutical compositions) containing cytoplasm are also provided. In some embodiments, the combination The product is formulated for different routes of administration (e.g., intravenous, subcutaneous, intramuscular, posterior orbital, intraperitoneal). In some embodiments, the composition is provided with a pharmaceutically acceptable carrier (e.g., phosphate phosphate). May contain (physiological saline).

[0101] Regarding the systemic administration of therapeutic cells, two major factors are crucial for successful homing of them to affected tissue. There is a big problem. Firstly, most cells are trapped in tiny capillaries in the lungs or other tissues. This could potentially lead to serious side effects, such as pulmonary embolism. The cytoplasm is, in some embodiments, much smaller than the parent cell (for example, the parent cell) (Approximately 60% of the diameter and 1 / 8 the volume of the parent cell), and lacking a rigid nucleus, the cytoplasm is smaller than that of the parent cell. It can also pass through small capillaries and blood vessels well. Secondly, cells reach the affected tissue. Specific homing is used in chemokine receptor signaling, e.g., SDF-1a / CXCR 4. It may depend on CCL2 / CCR2 and adhesive molecules, such as PSGL-1. As shown, the cytoplasm is manipulated to produce functional CXCR4, CCR2, and glycosyl It is possible to specifically express chemical PSGL-1, thereby enabling the specificity of the cytoplasm manipulated by it. Target homing can be significantly enhanced.

[0102] In some embodiments, the cytoplasm is a target expressed on the cell surface of the cytoplasm. The ing part, for example, CXCR4, CCR2 or PSGL-1 (for example, by operation, Or they may further include (from the cells from which they were obtained). Expression on the cell surface of the cytoplasm. A non-limiting example of a cell surface protein that could be a chemokine is CXCR4. Examples include CCR2, CCR1, CCR5, CXCR7, CXCR2, and CXCR1. In some embodiments, the cytoplasm is secreted by the cytoplasm, or by the cell Cell targeting region anchored to the outer matrix, e.g., SDFla or CCL2. Further components can be included (for example, by manipulation or from the cells from which they are obtained). As a non-limiting example of proteins that can be secreted by the cytoplasm for cell homing, So, SDFla, CCL2, CCL3, CCL5, CCL8, CCL1, CXCL9, Examples include CXCLIO, CCL11, and CXCL12. In some embodiments, The cytoplasm has surface markers that help evade the target immune system (for example, by manipulation). These may further include (from the cells from which they were obtained). For example, in some embodiments The cytoplasm may contain the CD47 marker. It is not bound by any particular theory. However, the CD47 marker prevents cytoplasm from being phagocytosed by macrophages. This is thought to be helpful in the non-limited interaction of cell matrix receptors and intercellular adhesion molecules. Examples include integrins, cadherins, glycoproteins, and heparin sulfate proteoproteins. Glycans are one example. Non-limiting examples of therapeutic molecules include tumor antigens and immunomodulatory compounds. Examples include butylenes, polyamines, and ATP.

[0103] In some embodiments, the cytoplasm lasts for about 1 to 7 days (for example, about 1 to 6 days) Approximately 1 to 5 days, approximately 1 to 4 days, approximately 1 to 3 days, approximately 1 to 2 days, approximately 2 to 7 days Approximately 2 to 6 days, approximately 2 to 5 days, approximately 2 to 4 days, approximately 2 to 3 days, approximately 3 to 7 days, Approximately 3 to 6 days, approximately 3 to 5 days, approximately 3 to 4 days, approximately 4 to 7 days, approximately 4 to 6 days, Approximately 4 to 5 days, approximately 5 to 7 days, approximately 5 to 6 days, or between approximately 6 and 7 days), approximately - 80℃ to approximately 16℃ (for example, approximately -80℃ to approximately 12℃, -80℃ to approximately 10℃, approximately -80℃ to approximately 16℃) Approximately 8°C, approximately -80°C to approximately 6°C, approximately -80°C to approximately 4°C, approximately -80°C to approximately 2°C, approximately -80°C to Approximately 0°C, approximately -80°C to approximately -4°C, approximately -80°C to approximately -10°C, approximately -80°C to approximately -16°C, approximately -80℃ to approximately -20℃, approximately -80℃ to approximately -25℃, approximately -80℃ to approximately -30℃, approximately -80℃ ~approximately -35℃, approximately -80℃~approximately -40℃, approximately -80℃~approximately -45℃, approximately -80℃~approximately -5 0℃, approximately -80℃ to approximately -55℃, approximately -80℃ to approximately -60℃, approximately -80℃ to approximately -65℃, approximately -80℃ to approximately -70℃, approximately -60℃ to approximately 16℃, approximately -60℃ to approximately 12℃, approximately -60℃ to approximately 10℃, approximately -60℃ to approximately 8℃, approximately -60℃ to approximately 6℃, approximately -60℃ to approximately 4℃, approximately -60℃ to Approximately 2°C, approximately -60°C to approximately 0°C, approximately -60°C to approximately -4°C, approximately -60°C to approximately -10°C, approximately -6 0℃ to approximately -10℃, approximately -60℃ to approximately -16℃, approximately -60℃ to approximately -20℃, approximately -60℃ to approximately -25℃, approximately -60℃ to approximately -30℃, approximately -60℃ to approximately -35℃, approximately -60℃ to approximately -40℃ Approximately -60°C to approximately -50°C, approximately -50°C to approximately 16°C, approximately -50°C to approximately 12°C, approximately -50°C ~10℃, approx. -50℃~approx. 8℃, approx. -50℃~approx. 6℃, approx. -50℃~approx. 4℃, approx. -50 ℃ to approximately 2℃, approximately -50℃ to approximately 0℃, approximately -50℃ to approximately -4℃, approximately -50℃ to approximately -10℃, approximately -50℃ to approximately -16℃, approximately -50℃ to approximately -20℃, approximately -50℃ to approximately -30℃, approximately -50℃ ~approximately -40℃, approximately -20℃ to approximately 16℃, approximately -20℃ to approximately 12℃, approximately -20℃ to approximately 10℃, Approximately -20°C to 8°C, approximately -20°C to 6°C, approximately -20°C to 4°C, approximately -20°C to 2°C. -20°C to 0°C, approx. -20°C to approx. -4°C, approx. -20°C to approx. -10°C, approx. -20°C to approx. -15℃, approximately -10℃ to approximately 16℃, approximately -10℃ to approximately 12℃, approximately -10℃ to approximately 10℃, approximately - 10℃ to approximately 8℃, approximately -10℃ to approximately 6℃, approximately -10℃ to approximately 4℃, approximately -10℃ to approximately 2℃, approximately - 10℃ to approximately 0℃, approximately -10℃ to approximately -4℃, approximately -10℃ to approximately -6℃, approximately -4℃ to approximately 16℃, Approximately -4°C to 10°C, approximately -4°C to 6°C, approximately -4°C to 4°C, approximately -4°C to 2°C, approximately -4 ℃ to approximately 0℃, approximately -2℃ to approximately 16℃, approximately -2℃ to approximately 12℃, approximately -2℃ to approximately 10℃, approximately -2℃ ~6°C, approx. -2°C to approx. 4°C, approx. -2°C to approx. 2°C, approx. -2°C to approx. 0°C, approx. 0°C to approx. 16°C Approximately 0°C to 14°C, approximately 0°C to 12°C, approximately 0°C to 10°C, approximately 0°C to 8°C, approximately 0°C to Approximately 6°C, approximately 0°C to approximately 4°C, approximately 2°C to approximately 16°C, approximately 2°C to approximately 12°C, approximately 2°C to approximately 10°C, approximately 2°C to approximately 8°C, approximately 2°C to approximately 6°C, approximately 2°C to approximately 4°C, approximately 4°C to approximately 16°C, approximately 4°C to approximately 12°C Approximately 4°C to 10°C, approximately 4°C to 8°C, approximately 4°C to 6°C, approximately 6°C to 16°C, approximately 6°C to 8°C 12℃, approximately 6℃ to approximately 10℃, approximately 6℃ to approximately 8℃, approximately 8℃ to approximately 16℃, approximately 8℃ to approximately 12℃, approximately 8°C to approximately 10°C, approximately 10°C to approximately 16°C, approximately 10°C to approximately 12°C, or approximately 12°C to approximately 16°C It can be stored at the temperature of ).

[0104] Furthermore, this specification also provides kits containing any of the compositions described herein. For example The kit includes instructions for using any of the compositions or methods described herein. May include. In some embodiments, the kit contains a small amount of any of the compositions described herein. It may contain at least one dose.

[0105] Several embodiments have been described. However, various modifications are intended to capture the spirit and scope of the present invention. Please understand that this can be done without deviating from the established framework.

[0106] Exemplary embodiments: Embodiment 1 includes a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, and a therapeutic peptide. One or more selected from the group consisting of cytotoxic drugs, small molecule therapeutics, and therapeutic gene editing factors. A therapeutically effective dose of a composition containing a first cytoplasm expressing or containing a number of molecules is administered. This is a method that includes the step of doing so.

[0107] Embodiment 2 is a first cytoplasm that is a mammalian cell, a protist cell, an algal cell, a plant cell, From fungal cells, invertebrate cells, fish cells, amphibian cells, reptile cells, or bird cells This is the method according to Embodiment 1, obtained from cells selected from the group.

[0108] Embodiment 3 is an embodiment in which the cells are cells taken from or derived from the subject. This is the method described in Form 2.

[0109] Embodiment 4 is a method in which the cells are cell lines, immortalized cells, or cancer cells, or are similar to them. This is a method derived from any one of Embodiments 2 to 3.

[0110] Embodiment 5 is any of Embodiments 1 to 4, wherein the first cytoplasm is obtained from immune cells. One method is as follows.

[0111] Embodiment 6 is a first cytoplasm consisting of natural killer (NK) cells, neutrophils, macrophages. A group selected from phagocytes, eosinophils, basophils, dendritic cells, and lymphocytes. The method is obtained from cells according to any one of Embodiments 1 to 5.

[0112] Embodiment 7 is characterized in that the first cytoplasm consists of hematopoietic stem cells, mammary gland stem cells, intestinal stem cells, and mesenchymal stem cells. Endothelial stem cells, neural stem cells, olfactory adult stem cells, neural crest stem cells, skin stem cells, testicular cells, embryo Obtained from cells selected from the group consisting of sex stem cells, fibroblasts, or induced pluripotent stem cells. The method is described in any one of Embodiments 1 to 4.

[0113] Embodiment 8 is a combination of Embodiments 1 to 7, in which the first cytoplasm is fused with the second cytoplasm. The method is one of the following.

[0114] Embodiment 9 is a second cell which is a mammalian cell, a protist cell, an algal cell, a plant cell, a true It consists of fungal cells, invertebrate cells, fish cells, amphibian cells, reptile cells, or avian cells. This is the method according to Embodiment 8, obtained from cells selected from the group.

[0115] Embodiment 10 is a therapeutic RNA molecule that is a messenger RNA (mRNA), short he Apin RNA (shRNA), small interfering RNA (siRNA), microRNA, long chain One of embodiments 1 to 9 is a non-coding RNA (IncRNA) or RNA virus. The method is one of the following.

[0116] Embodiment 11 is a therapeutic DNA molecule consisting of single-stranded DNA, double-stranded DNA, and oligonucleotides. The DNA molecules, plasmids, bacterial DNA molecules, or DNA viruses of embodiments 1 to 10 The method is one of the following.

[0117] Embodiment 12 describes a therapeutic protein that is an enzyme, antibody, antigen, toxin, cytokine, or protein. Embodiments 1 to 11 are either cytoplasmic hormones, growth factors, cell surface receptors, or vaccines. The method is one of the following.

[0118] Embodiment 13 is a cytoplasm containing therapeutic DNA molecules, therapeutic RNA molecules, and therapeutic proteins. transient expression of therapeutic peptides, small molecule therapeutics, and / or therapeutic gene editing factors This is a method according to any one of embodiments 1 to 12.

[0119] Embodiment 14 includes a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, and a therapeutic pe The expression of plutides, small molecule therapeutics, and / or therapeutic gene editing factors can be induced. The method is described in any one of Embodiments 1 to 12.

[0120] Embodiment 15 is a peptide therapeutic agent selected from the group consisting of peptide hormones and antigens. The method is described in any one of Embodiments 1 to 14.

[0121] Embodiment 16 describes small molecule therapeutics as steroids, polyketides, alkaloids, toxins, and antibiotics. Substances, antiviral drugs, analgesics, anticoagulants, antidepressants, anticancer drugs, antiepileptic drugs, antipsychotics Drugs, sedatives, colchicine, taxol, mitomycin, emtansine, or currently available Embodiment 1 is selected from the group consisting of any possible or developing small molecule therapeutics. The method is one of the methods described in 15.

[0122] Embodiment 17 is an embodiment in which the cytoplasm contains small molecule therapeutic agents or therapeutic nanoparticles. The method is one of the methods described in 1 through 16.

[0123] Embodiment 18 is a cytoplasm of bacteria, bacterial spores, bacteriophages, bacterial components, and viruses. The therapeutic agent contains a substance selected from the group consisting of ions, exosomes, lipids, and ions. The method is one of the methods described in any one of the methods 1 to 16.

[0124] Embodiment 19 is the method of Embodiment 18, wherein the virus is an oncolytic virus. be.

[0125] Embodiment 20 is a group of small molecule therapeutic agents consisting of anticancer agents, antibiotics, or antiviral agents. A method according to any one of embodiments 1 to 15 or 17 to 19, selected from be.

[0126] Embodiment 21 further includes a step of applying to one or more additional therapies. The method is one of the methods described in any one of the methods 1 to 19.

[0127] Embodiment 22 is an embodiment in which one or more additional therapies are cell-based therapies, small molecule therapies, or immunotherapies. The treatment is selected from a group consisting of chemotherapy, radiotherapy, gene therapy, and surgery. This is the method described in Form 20.

[0128] Embodiment 23 is a configuration of Embodiments 1 to 21 in which the first cytoplasm expresses an immune system evasion region. The method is one of the following.

[0129] Embodiment 24 is the method described in Embodiment 23, wherein the immune system evasion portion is CD47. .

[0130] Embodiment 25 is a cell from which the first cytoplasm or the cell from which the first cytoplasm is obtained contains therapeutic DNA Molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, non-peptide therapeutics, and Embodiments 1 to 24 are engineered to express and / or therapeutic gene editing factors. The method is one of the following.

[0131] Embodiment 26 is a cell from which the first cytoplasm or the cell from which the first cytoplasm is obtained contains therapeutic DNA Molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, non-peptide therapeutics, and Not modified to express either a and / or therapeutic gene editing factor, The method is one of the methods described in any one of states 1 to 24.

[0132] Embodiment 27 is a further embodiment of Embodiments 1 to 26 in which the composition further includes a targeting portion. The method is one of the following.

[0133] Embodiment 28 is described in Embodiment 27, wherein the targeting portion is a cell surface protein. This is the method of installation.

[0134] Embodiment 29 is an embodiment in which the targeting portion targets secreted proteins or extracellular matrix. This is the method according to Embodiment 27, in which the protein is tethered.

[0135] Embodiment 30 is a further embodiment of Embodiments 1 to 26, wherein the cytoplasm further includes a targeting portion. The method is one of the following.

[0136] Embodiment 31 is described in Embodiment 30, wherein the targeting portion is a cell surface protein. This is the method of installation.

[0137] Embodiment 32 is an embodiment in which the targeting portion targets secreted proteins or extracellular matrix. The method according to Embodiment 30, wherein the protein is tethered.

[0138] Embodiment 33 is a cytoplasm containing at least one therapeutic agent.

[0139] Embodiment 34 is a therapeutic agent comprising a therapeutic DNA molecule, a therapeutic RNA molecule, a therapeutic protein, A therapeutic peptide, a small molecule therapeutic agent, or a therapeutic gene editing factor, as described in Embodiment 33. It is the cytoplasm of the organism.

[0140] Embodiment 35 is a therapeutic RNA molecule that is a messenger RNA (mRNA), short he Apin RNA (shRNA), small interfering RNA (siRNA), microRNA, long chain The details described in Embodiment 34, which are non-coding RNA (IncRNA) or RNA viruses. It is a cytoplasmic body.

[0141] Embodiment 36 is a therapeutic DNA molecule consisting of single-stranded DNA, double-stranded DNA, and oligonucleotides. Embodiments 34 to 35 are plasmids, bacterial DNA molecules, or DNA viruses. It is a cytoplasm described in any one of the following.

[0142] Embodiment 37 describes a therapeutic protein that is an enzyme, antibody, antigen, toxin, cytokine, or protein. Embodiments 34 to 3 are cytohormones, growth factors, cell surface receptors, or vaccines. It is a cytoplasm described in any one of the six items.

[0143] Embodiment 38 is a peptide therapeutic agent selected from the group consisting of peptide hormones and antigens. The cytoplasm is as described in any one of embodiments 34 to 37.

[0144] Embodiment 39 describes small molecule therapeutic agents as steroids, polyketides, alkaloids, toxins, and antibiotics. Substances, antiviral drugs, analgesics, anticoagulants, antidepressants, anticancer drugs, antiepileptic drugs, antipsychotics Drugs, sedatives, colchicine, taxol, mitomycin, emtansine, or currently available Embodiment 34 is selected from the group consisting of any possible or developing small molecule therapeutics. It is a cytoplasmic body described in any one of the 38 items from to 38.

[0145] Embodiment 40 is a therapeutic agent comprising nanoparticles, bacteria, bacterial spores, bacteriophages, and bacterial compounds. Embodiments selected from the group consisting of molecules, viruses, exosomes, lipids, and ions. It is a cytoplasm described in any one of 33 to 38.

[0146] Embodiment 41 is the cytoplasm described in Embodiment 40, wherein the virus is an oncolytic virus. It is the body.

[0147] Embodiment 42 is a group of small molecule therapeutic agents consisting of anticancer agents, antibiotics, or antiviral agents. A cytoplasm according to any one of embodiments 33 to 41, selected from the above.

[0148] Embodiment 43 is a further embodiment of Embodiments 33 to 43 in which the cytoplasm further includes an immune system evasion portion. It is the cytoplasm described in either one of the following.

[0149] Embodiment 44 is the cytoplasm described in Embodiment 43, wherein the immune system evasion portion is CD47. be.

[0150] Embodiment 45 is, Therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutics Introducing therapeutic drugs, therapeutic gene editing factors, other therapeutic agents, and / or therapeutic nanoparticles into cells. The steps to take; The step of enucleating the cells and This is a method for producing cytoplasm that includes [a specific component].

[0151] Embodiment 46 is described in Embodiment 45, wherein the introduction step precedes the denucleation step. This is the method of installation.

[0152] Embodiment 47 is an introduction step in which a therapeutic DNA molecule, a therapeutic RNA molecule, and a therapeutic Permanent proteins, therapeutic peptides, small molecule therapeutics, and / or therapeutic gene editing factors This is the method according to Embodiment 46, which results in continuous expression.

[0153] Embodiment 48 is described in Embodiment 45, wherein the denucleation step precedes the introduction step. This is the method of installation.

[0154] Embodiment 49 is an introduction step in which therapeutic DNA molecule, therapeutic RNA molecule, therapeutic Proteins, therapeutic peptides, small molecule therapeutics, and / or therapeutic gene editing factors In the method of any one of embodiments 45, 46, or 48, which results in excessive expression be.

[0155] Embodiment 50 is a therapeutic RNA molecule that is a messenger RNA (mRNA), short he Apin RNA (shRNA), small interfering RNA (siRNA), microRNA, long chain Embodiments 45 to 49 are non-coding RNA (IncRNA) or RNA viruses. The method is one of the following.

[0156] Embodiment 51 is a therapeutic DNA molecule consisting of single-stranded DNA, double-stranded DNA, and oligonucleotides. Embodiments 45 to 50 are plasmids, bacterial DNA molecules, or DNA viruses. The method is one of the following.

[0157] Embodiment 52 describes a therapeutic protein that is an enzyme, antibody, antigen, toxin, cytokine, or protein. Embodiments 45 to 5 are cytohormones, growth factors, cell surface receptors, or vaccines. The method is one of the methods described in 1.

[0158] Embodiment 53 is a peptide therapeutic agent selected from the group consisting of peptide hormones and antigens. The present invention relates to the method described in any one of embodiments 45 to 52.

[0159] Embodiment 54 describes small molecule therapeutic agents as steroids, polyketides, alkaloids, toxins, and antibiotics. Substances, antiviral drugs, analgesics, anticoagulants, antidepressants, anticancer drugs, antiepileptic drugs, antipsychotics Drugs, sedatives, colchicine, taxol, mitomycin, emtansine, or currently available Embodiment 45 is selected from the group consisting of any possible or developing small molecule therapeutics. The method is one of the methods described in 53.

[0160] Embodiment 55 is a further embodiment of Embodiments 45 to 54, wherein the cytoplasm further comprises therapeutic nanoparticles. The method is one of the following.

[0161] Embodiment 56 is a cytoplasm of bacteria, bacterial spores, bacteriophages, bacterial components, and viruses. The therapeutic agent further comprises a selection from the group consisting of s, exosomes, lipids, and ions. This is a method according to any one of embodiments 45 to 54.

[0162] Embodiment 57 is the method of Embodiment 56, wherein the virus is an oncolytic virus. be.

[0163] Embodiment 58 is derived from Embodiment 45, wherein the introduction step includes transfection. The method is one of the 57 methods.

[0164] Embodiment 59 is an introduction step where electroporation, microinjection Cell squeezing, sonoporation, impalement, or fluid force The method is according to any one of embodiments 45 to 58, including academic delivery.

[0165] Embodiment 60 is, The step of transfecting cells with a vector; The step of enucleating the cells and This is a method for producing cytoplasm that includes [a specific component].

[0166] Embodiment 61 is an implementation in which the transfecting step precedes the denucleation step. This is the method described in form 60.

[0167] Embodiment 62 is performed after the enucleation step has been incorporated into the cell genome. This is the method described in Embodiment 61.

[0168] Embodiment 63 is an embodiment in which the denucleation step precedes the transfecting step. This is the method described in form 60.

[0169] Embodiment 64 is any of Embodiments 60 to 63, wherein the vector is a viral vector. One method is to use this method.

[0170] Embodiment 65 is a viral vector that is a retroviral vector, adeno-associated virus ( AAV vector, vesicular virus vector, or hybrid virus vector. This is the method described in Embodiment 64.

[0171] Embodiment 66 is derived from Embodiment 60, wherein the vector contains a coding sequence for a therapeutic protein. The method is one of the 65 methods.

[0172] Embodiment 67 describes a therapeutic protein that is an enzyme, antibody, antigen, toxin, cytokine, or protein. Embodiment 66 describes a cytoplasmic hormone, growth factor, cell surface receptor, or vaccine. This is the method.

[0173] Embodiment 68 is a method for producing a cytoplasm, which includes the step of enucleating a cell.

[0174] Embodiment 69 is the method of Embodiment 68, wherein the cells are not erythroblasts.

[0175] Embodiment 70 is an embodiment in which the denucleation step includes centrifugation, similar to Embodiment 68 or Embodiment 6. This is the method described in 9.

[0176] Embodiment 71 is a therapeutically effective cytoplasm according to any one of Embodiments 33 to 44. This is a method of treating a target, which includes the step of administering a specific amount to the target.

[0177] Embodiment 72 is a method comprising the step of administering a therapeutically effective amount of cytoplasm to a target. .

[0178] Embodiment 73 is the same as Embodiment 72, but the cytoplasm is not obtained from erythroblasts. It is a method.

[0179] Embodiment 74 is, Steps to produce cytoplasm by the method described in any one of Embodiments 45 to 70. and; Steps to preserve the cytoplasm and This method includes [something].

[0180] Embodiment 75 is the method of Embodiment 74, wherein the preservation step includes cryopreservation. ru.

[0181] Embodiment 76 is the method of Embodiment 74, wherein the preservation step includes cryopreservation. ru.

[0182] Embodiment 77 is, The step of culturing cells in a culture medium; Steps to stimulate cells; The steps of enucleating cells and forming cytoplasm and This method includes [something].

[0183] Embodiment 78 is a culture step in which the culture steps include 3D culture, adherent culture, suspension culture, and semi-suspension culture. The method according to Embodiment 77, comprising one or more of the nutrients.

[0184] Embodiment 79 is a step in which the cells are stimulated by adding one or more drugs to the culture medium. Adding one or more antibodies to the culture medium, culturing one or more exosomes Adding to the substrate, adding one or more chemokines to the culture medium, one or more Adding the cytoplasm to the culture medium, culturing under 2D or 3D conditions, or using low acidity Any of embodiments 77 to 78, which includes one or more of culturing under basic conditions. One method is to use this method.

[0185] Embodiment 80 further includes the step of separating cells or cytoplasm from the culture medium. The method is described in any one of forms 77 to 80.

[0186] Embodiment 81 is, The step of culturing cells in a culture medium; Steps to stimulate cells and A method including, A method comprising the step of stimulating cells by adding one or more cytoplasmic cells to a culture medium. That is the case.

[0187] Embodiment 82 is a culture step in which the culture steps include 3D culture, adherent culture, suspension culture, and semi-suspension culture. The method according to Embodiment 81, comprising one or more of the nutrients.

[0188] Embodiment 83 is a cell stimulating step in which one or more drugs are added to the culture medium. Adding one or more antibodies to the culture medium, culturing one or more exosomes To add to the substrate, to add one or more chemokines to the culture medium, 2D or 3 Cultivating under conditions D, or cultivating under hypoxic conditions, one or more of these. Furthermore, the method according to any one of embodiments 81 to 82 is also included.

[0189] Embodiment 84 further includes the step of separating the cells from the culture medium, as described in Embodiments 81 to 8. The method is one of the three methods.

[0190] Embodiment 85 further includes the step of enucleating cells to form cytoplasm, The method is one of the methods described in 81 to 84.

[0191] Embodiment 86 is a culture prepared by the method described in Embodiment 80 or Embodiment 84. A method for treating a subject, comprising the step of administering a therapeutically effective dose to the subject.

[0192] Embodiment 87 is a cell prepared by the method described in Embodiment 80 or Embodiment 86. This is a method of treating a subject, which includes the step of administering a therapeutically effective dose of the substance to the subject.

[0193] Embodiment 88 is for cancer, infectious diseases, neurological diseases, degenerative diseases, autoimmune diseases, cardiovascular diseases, and eye diseases. In the manufacture of pharmaceuticals for the treatment of vascular diseases, bone diseases, metabolic diseases, or two or more of these. This is the use of the cytoplasm as described in any one of embodiments 33 to 44.

[0194] Embodiment 89 is for cancer, infectious diseases, neurological diseases, degenerative diseases, autoimmune diseases, cardiovascular diseases, and eye diseases. In the manufacture of pharmaceuticals for the treatment of vascular diseases, bone diseases, metabolic diseases, or two or more of these. This involves using a culture medium prepared by the method of Embodiment 80 or Embodiment 84.

[0195] Embodiment 90 includes the step of administering to the cytoplasm, for the disease or This is a method for determining whether or not a state exists.

[0196] Embodiment 91 is a disease or condition in which cancer, infectious disease, inflammatory condition, neurological disease, degenerative disease, self Autoimmune diseases, cardiovascular diseases, ischemic diseases, hereditary or genetic conditions, developmental conditions, ophthalmic diseases Embodiments include bone diseases, metabolic diseases, poisoning, idiopathic diseases, or two or more of these. This is the method described in 90.

[0197] Embodiment 92 is an embodiment in which the cytoplasm expresses or contains a reporter molecule or reagent. The method is one of the methods described in states 90 to 91.

[0198] Embodiment 93 is a bioreporter molecule or reagent. This is the method described in Embodiment 92.

[0199] Embodiment 94 is, A first cytoplasm which is a cytoplasm according to any one of embodiments 33 to 44; The cell or second cytoplasm and It is a cell fusion product that includes [the specified element].

[0200] Embodiment 95 is, Steps to obtain a sample from the subject; The step of adding cytoplasm to the sample and This method includes determining the presence or absence of disease or condition in a subject.

[0201] Embodiment 96 describes a disease or condition such as cancer, infectious disease, inflammatory condition, neurological disease, degenerative disease, or autoimmune disease. Autoimmune diseases, cardiovascular diseases, ischemic diseases, hereditary or genetic conditions, developmental conditions, ophthalmic diseases Embodiments include bone diseases, metabolic diseases, poisoning, idiopathic diseases, or two or more of these. This is the method described in 95.

[0202] Embodiment 97 is an embodiment in which the cytoplasm expresses or contains a reporter molecule or reagent. The method is one of the methods described in states 95 to 96.

[0203] Embodiment 98 is a bioreporter molecule or reagent. This is the method described in Embodiment 97. [Examples]

[0204] This disclosure will be further illustrated by the following embodiments, which are described in the claims. This does not limit the scope of disclosure.

[0205] [Example 1] - Successful enucleation and survival of mammalian cells As shown in Figure 1, therapeutic cytoplasm is generated from cells of the same species or from the patient's own donor. It can be used for the treatment and diagnosis of diseases. As a proof of concept, various types Mammalian cells of the genus (e.g., mesenchymal stem cells, neutrophils, fibroblasts, natural killer cells) The enucleation efficiency and recovery rate of cells were determined. Mammalian cells were removed from cell culture plates. Afterward, mammals were identified by density gradient centrifugation using discontinuous Ficoll gradient high-speed centrifugation. The cells were enucleated (Figures 2A-D). Table 1 summarizes the results of enucleation using the suspension protocol. Enucleation efficiency and cell viability are observed in hTERT-transformed cells and primary mesenchymal stem cells. The levels were highest in both (MSCs), as well as in fibroblasts and neutrophils. Table 2 shows adhesion This report summarizes the results of enucleation using Rotocall. Enucleation efficiency is measured for mesenchymal stem cells and macromolecules. Both lophages showed over 70%. This experiment was conducted using any of the methods described herein. We demonstrated that various types of mammalian cells can be enucleated using this method.

[0206] [Table 1]

[0207] [Table 2]

[0208] Next, the cytoplasmic viability was measured over 96 hours (Figure 3A). MSCs proliferated over time. However, the cytoplasm did not proliferate. Instead, there were relative changes in the viable cytoplasm. The magnification remained appropriately constant for 72 hours until it decreased at 96 hours. Therefore, the cytoplasm The body survived for 3-4 days. Most cell-based therapies can be used immediately. Since there was no data available, we determined the survival rate of the cytoplasm after cryopreservation. Surprisingly, the cells after cryopreservation... The survival rate of the cells was higher than that of MSCs after cryopreservation (Figure 3B). Seeding occurred immediately after enucleation. Seeded cytoplasm and cytoplasm recovered from cryopreservation showed similar relative performance after 24 hours. The cell viability was shown (Figure 3C). This experiment showed that cytoplasmic viability was not affected by cryopreservation. This demonstrated that the survival rate of cytoplasm after cryohibernation was the survival rate of MSCs after cryohibernation. The rates were similar (Figure 25A). The cytoplasm recovered after cryopreservation of varying lengths was Similar to MSCs recovered after cryopreservation, they were induced by the Boyden chamber assay. It was able to receive the movement (Figure 25B).

[0209] Next, large-scale cell production is set up ex vivo, followed by high-volume density gradient centrifugation. Enucleation was performed to generate therapeutic cytoplasm. In one embodiment, the therapeutic cytoplasm was used to treat disease A therapeutic cargo (e.g., mRNA, drugs, peptides, etc.) for treating the condition is loaded. In another embodiment, therapeutic cytoplasm is used for immediate use in diagnosis. Prepared (e.g., intravenous injection (IV), intraperitoneal injection (IP), tissue, or in v (Applicable to ITR)

[0210] [Example 2] - The cytoplasm retains the ability to interact with intact organelles and the extracellular matrix, Cells can perform biological functions and function as therapeutically valuable delivery vehicles. After determining whether the cytoplasm can maintain its viability after cryopreservation, the cytoplasm derived from MSCs is then selected. To determine whether the cell surface marker profile of the body differs from that of bone marrow-derived MSCs Flow cytometry analysis was performed (Figure 4). As shown in Figure 4, the fine particles derived from MSCs were observed. Both cystoid and bone marrow-derived MSCs are CD45, CD90, CD44, CD146. and maintained cell surface expression of CD166. Figures 5A-F' and 6A-D' show cells. The matrix proteins attach, reorganize the cytoskeleton, and modify the matrix proteins in 2D and 3D culture systems. They spread across the substrate, forming tunnel nanotubes, which are cells of the same or different origins. It was shown that biological products can be transferred between them. Organelle staining was performed on the Golgi, ER, and F-acti. The cytoskeleton, lysosomes, endosomes, microtubules, and mitochondria are located within the cytoplasm. It was shown to remain intact (Figure 7A-E'). Furthermore, the cytoplasm was shown in vitro. This demonstrated the possibility of homing at o. The cytoplasm is contained on extracellular matrix proteins. They moved easily and moved in the direction of the soluble chemokine gradient (i.e., via chemosensibility). (Figures 8A and 8B). In particular, cytoplasm exogenously transfected with purified mRNA. It generates functional intracellular proteins and develops them for various clinical uses and disease conditions. This can mimic the therapeutic mRNA application that has been performed. Furthermore, this can mimic mRNA translation and protein The mechanisms related to plasmogenesis function normally in the cytoplasm in the absence of the nucleus, thus having therapeutic value. It has been demonstrated that it can be used to generate certain bioactive molecules.

[0211] Cells exogenously transfected with purified mRNA encoding a known secretory protein. The substance generates functional extracellular proteins in conditioned culture medium, and this is related to the ER / Golgi and This demonstrates that the secretory pathway functions normally in the cytoplasm in the absence of the nucleus (Figure 11). Furthermore, macrophages and endothelium were conditioned using a cytoplasmic conditioning medium containing secreted proteins. Treatment of these cells activated important signaling responses in them (Figure 12). This allows the cytoplasm to produce and deliver therapeutically valuable secreted proteins and biomolecules. A proof of concept was provided that it can be used as a novel vehicle to reach [the target]. While not limited to siRNA, shRNA, mRNA, DNA plasmids, and PEG-12 nucleotides, this does not limit the types of nucleotides that can be nucleotides. It can be loaded with various cargoes, including butylene and chemotherapeutic agents (for example, Figure 9). See also section 10).

[0212] [Example 3] - Manipulated cytoplasm can function both in vitro and in vivo. While I don't want to be constrained by theory, this example illustrates the concept of "cargo," for example, outside This demonstrates that cytoplasms can be generated that are manipulated to express the causative mRNA molecule. Figures 13B and 13C show manipulation of MSC-derived cytoplasm to achieve therapeutic-level functional anti-inflammatory effects. Sexual cytokine interleukin-10 (IL-10) in vitro and intravenously This demonstrates that it can be generated and secreted in a preclinical mouse model after internal injection. Figure 13B shows: Cytoplasm transfected with IL-10 mRNA secretes high levels of IL-10. This indicates that it is obtained. To determine whether the secreted IL-10 is active, blood Starvated macrophages, untreated MSCs, MSCs expressing IL-10, untreated cytoplasm and incubated with cytoplasm expressing IL-10 in conditioned medium (CM). Phosphorylated STAT3 was incubated with CM of MSCs expressing IL-10. After that, and after incubation with CM of cytoplasm expressing IL-10, macro Detected by phage, but CM and incubate from untreated MSCs and untreated cytoplasm. STAT3 activity was not detected in macrophages after vaping (Figure 13C). To determine whether IL-10 secreted from the cytoplasm is detectable in vivo. Therefore, C57B1 / 6 mice were given MSCs or MSC-derived cytoplasm that express IL-10. The drug was injected into the posterior orbit. Blood was collected two hours after the injection to determine the IL-10 level. In the blood of mice injected with untreated MSCs, IL-10 was little to no detectable. This was not the case (Figure 13D). As shown in Figure 13D, mice injected with untreated MSCs Compared to the levels found in other sources, higher levels of IL-10 are found in MSCs that express IL-10. It was detected in mice injected with cytoplasm.

[0213] These data are clinically relevant for therapeutic use in treating normal and affected tissues. Potential of genetically engineered cytoplasm-based cell therapies that generate and secrete cytokines This indicates that.

[0214] To determine whether MSC-derived cytoplasm can penetrate the basement membrane, MSC or MSC The derived cytoplasm was allowed to penetrate the basement membrane toward 10% FBS for 24 hours. Figures 14A and 1 As shown in 4B, MSC-derived cytoplasm was compared with untreated MSCs in the presence of 10% FBS. They were equally efficient at penetrating the basement membrane. Notably, untreated MSCs Although they were able to penetrate the basement membrane in the absence of chemottractants, the cytoplasm of MSCs treated with chemotactic agents transformed. In the absence of attractants, penetration into the basement membrane was almost impossible. These data This indicates that MSC-derived cytoplasm can digest the basement membrane and enter it. The data shows that cytoplasmic cell therapies can transcend complex extracellular matrix barriers. It demonstrates the unique potential to move and deliver those cargoes(s) within the organization.

[0215] As shown in Figures 15A and 15B, MSC-derived cytoplasm has an average diameter of 12 μm. However, MSCs have an average diameter of 20 μm. Determining the in vivo distribution of MSC-derived cytoplasm. To achieve this, mice were injected posteriorly into the orbit of MSCs or MSC-derived cytoplasm. Figure 15C As shown in 15D, the number of MSC-derived cytoplasmic cells was greater than the number of MSCs detected in the liver. It was detected in the liver. These data are cytoplasmic-based molecules that are directly delivered into circulation. Blister therapy has shown potential to treat a wide range of diseases.

[0216] [Example 4] - Manipulated cytoplasm can express functional cell surface proteins. As shown in Figure 16B, manipulated MSCs expressing CXCR4 and CXCR4 The manipulated MSC-derived cytoplasm that is represented is equivalent to that determined by flow cytometry. The manipulated cytoplasm expresses CXCR4 at a certain level. The manipulated cytoplasm expresses functional cell surface proteins. To determine whether or not it can be expressed, we used MSCs expressing the CXCR4 receptor and MSC-derived cells. The cytoplasm was moved towards SDF-1α at various concentrations. As shown in Figure 16C, the functional MSC-derived cytoplasm that has been engineered to express CXCR4 migrates toward SDF-1α. This can be achieved, and cell migration increases with increasing SDF-1α concentration. Furthermore, migration The number of MSC-derived cytoplasms was greater than the number of migrating MSCs expressing CXCR4. (Figure 16C).

[0217] Figures 17A-C show manipulation of MSC-derived cytoplasm to promote cell adhesion to the inflamed vascular system. It is possible to express functional cell adhesion proteins that are known to mediate this process. Figures 18A-D show the manipulation of MSC-derived cytoplasm to promote macrophage interactions. and express cellular proteins known to modulate the phagocytic activity of therapeutic cells. This indicates that it is possible.

[0218] [Example 5] -By manipulating the cytoplasm, it is possible to induce the secretion of functional IL-12, and also in related breast cancers. In this model, it is possible to induce the expression of inflammatory genes and suppress tumor growth. MSCs and MSC-derived cytoplasm were transfected with IL-12 mRNA. The culture medium (CM) was administered 24 hours, 48 ​​hours, and 72 hours after transfection. They were collected. As shown in Figure 19B, the cytoplasm derived from MSCs secretes IL-12. To determine whether MSC-derived cytoplasm can secrete functional IL-12, we used mouse spleen. Cells in complete culture medium, CM from MSCs expressing IL-12, MSCs expressing IL-12 MS expressed by IL-12 were treated with CM from the derived cytoplasm and purified IL-12. MSCs expressing C-derived cytoplasm and IL-12 are associated with STAT4 in mouse splenocytes. It secretes functional IL-12 that can cause phosphorylation (Figure 19C).

[0219] As shown in Example 3, post-orbital administration of cytoplasm was well tolerated in mice. To determine whether intratumoral administration of cytoplasmic cells is permissible, mice were given tumors located posterior orbital or tumors. The drug was administered by injection either intra-ulcer or intra-ulcer. The number of deaths was recorded and classified according to the injection method and cause of death. (Table 3). As shown in the table below, intratumoral administration of cytoplasmic bodies has an excellent safety profile. It was perfectly acceptable along with the file.

[0220] [Table 3]

[0221] Next, we established MSC-derived cytoplasms expressing IL-12 and empty MSC-derived cytoplasms. The E0771 subcutaneous tumor was injected. All mice were euthanized 48 hours after the injection. Then, the tumor sample was collected. As shown in Figure 19D, the tumor IL-12... It was detected in tumors isolated from mice injected with MSC-derived cytoplasm expressing the substance, In tumors isolated from mice injected with empty MSC-derived cytoplasm, tumor IL-12 These were hardly detected, or not detected at all. In summary, these results indicate that MSC-derived particles were not detected. The cytoplasm is present in the affected tissue in preclinical mouse models at a clinically relevant level for therapeutic use. This demonstrates the ability to produce, secrete, and deliver tokines.

[0222] Figures 20A-C show the injection of cytoplasm modified to express the IL-12 cytokine. Samples taken from mice containing interferon-gamma (IFNγ) and PD-L1 were found to be of high quality. And CXCL9 expressing, while being collected from mice that received PBS alone or empty cytoplasm. The samples showed low levels of IFNγ, PD-L1, and CXCL9. These data are from MSC-derived cytoplasm engineered to express IL-12. However, this indicates that it induced an inflammatory response within the injected tumor. Figure 20D shows IL-12 Injection of MSC-derived cytoplasm engineered to express [specific gene] showed a reduction in tumor size. It is.

[0223] Figures 21A-C show that oncolytic viruses can be loaded into MSC-derived cytoplasm, It is possible to deliver viruses like these to tumors that proliferate in immunodeficient and immunocompetent mice. This, combined with IL-12 secretion, leads to the infiltration of cytotoxic CD8+ T cells into tumors. This indicates that it promotes growth. Regarding Figure 21A, a very small number of cytoplasmic cells swell after 7 days. It can be detected in ulcers, but not in the center (injection site) and outer edges of growing tumors. It should be noted that there are numerous MSCs (Methodically Assured Stem Cells).

[0224] Figures 22A-B show that genetically modified MSC-derived cytoplasm delivers gene-editing proteins. Therefore, after cytoplasm-host cell fusion, gene function can be controlled in the host cell. This indicates that these data modify normal or mutant genes within cells. This demonstrates the potential of cytoplasm-based cell therapy that delivers editing components.

[0225] Ennucleation of mesenchymal stem cells (MSCs) This protocol is from Cell Biology Volume 14, 1976, Pages 87-93 Chapter 7 Enucleus. ation of Mammalian Cells in Suspension (Michael H. Wigler, Alfred I. Neugut, I. I modified Bernard Weinstein's method.

[0226] Preparation of 50% Ficoll solution: In a light-shielded glass beaker, add several grams of F icoll (PM400, GE Healthcare 17-0300-500) In milliliters of ultrapure water (Invitrogen 10977-015), at room temperature for 24 minutes. The mixture was dissolved by continuous magnetic stirring for several hours. Then, the mixture was autoclaved for 30 minutes. When the mixture cooled, it was stirred again to ensure a uniform viscosity. The refractive index was measured using a refractometer. When measured with (Reichert 13940000), the result was 1.4230~1.429 The values ​​were in the range of 0. The aliquots were stored at -20 degrees Celsius.

[0227] Preparation of 2X MEM: For each 50 ml volume, add 10 mL of 10X MEM (Gibco, 11430-030), more precisely 2.94 mL of sodium bicarbonate (7.5%, Gibc o, 25080-094), 1mL 100X Pen-Strep (Gibco 15 140-122) and 36 mL ultrapure water (Invitrogen 10977-015 ) was used. Then the solution was placed in a 0.22 μm membrane flask (Olympus 25-22). 7) It was filtered and stored at 4 degrees Celsius.

[0228] On the day before enucleation, MSCs were placed on a 15cm plate (Olympus 25-203) 2.5M, 20 mL MSC medium [MEM 1X (Gibco 12561-056)] ;16.5% Premium FBS (Atlanta Biologics SI 150) );1% HEPES 1M(Gibco 15630-80);1% Anti-An Gibco 15240-062 1% Glutamax 100X (Gibco 35050-061) was sown. Next, cytochalacin B (Sigm Aldrich C6762 was added to 2X MEM (final concentration 2 μM / mL). .

[0229] Preparation of Ficoll gradient: 2X CytoB to 50% Ficoll aliquot in a 1:1 ratio. Dilution was added to prepare a 25% Ficoll stock concentration. Next, 17%, 16%, and 1% concentrations were prepared. Add 5% and 12.5% ​​Ficoll to an appropriate amount of 1X MEM buffer (Cytokarashi Dilute 25% Ficoll by adding 2X MEM containing B to ultrapure water in a 1:1 dilution. It was prepared by the following method. The diluted solution was incubated in a CO2 incubator for at least 1 hour. The solution was then covered with a cap and equilibrated. Next, the Ficoll gradient was passed through 13.2 mL of ultra-clear tube. Pour into a piping bag (Beckman, 344059) and leave in a CO2 incubator overnight. Incubated for 6-18 hours.

[0230] On the day of enucleation, 12-25M MSC (ideally 20M) is injected into each tube for enucleation. The cells were collected. The culture medium was aspirated and the cells were phosphate-buffered saline (PBS) (GIBCO 141). Washed once with 90-144). 5 mL of TrypLE-Select (Gibco, 1 2563011) was added to each plate and incubated for up to 5 minutes. 90% of the cells were If isolated, add 5 mL of complete MSC medium and place the cells in a 50 mL tube (3-4 pegs). The mixture was collected in a rate / tube. The tube was then centrifuged at 1,200 rpm for 5 minutes. The pellet was resuspended in 10 mL of PBS. The cells were counted, pelletized, and 1 The cells were then resuspended in 2.5% Ficoll. Next, the cell-Ficoll mixture was filtered through a 40 μm cell filter. The solution was passed through a filter (Falcon 352340) and then dropped into a new 50 mL tube. Using a gradient, 3.2 mL of cell suspension was slowly loaded onto a pre-prepared gradient. 1 mL of 1X MEM buffer was added to the final (upper) layer using a syringe. Then, The tube is loaded into the rotor bucket, balanced, and then placed in the ultracentrifuge (Beckman). 60 minutes at L8M, 26,000 rpm, 31℃, Accel 7, Decel 7 The procedure was performed as follows. At the end of centrifugation, three layers were present: one was 12.5% ​​(cytoplasm and Near the top of the fragment, one is near the interface (cytoplasm) at 12.5 / 15%, and 25% The pellet (nucleus) at the bottom. Transfer the upper layer of 15% Ficoll solution to a 15 ml conical tube. The sample was collected. Next, the collected layer was diluted with more than 4 volumes of heated serum-free MSC medium. (i.e., filled with 3 mL of Ficoll and 15 mL or less of medium). Mix gently. After combining, the mixture was pelletized at 1,200 rpm for 10 minutes. The serum-free MSCs were then heated. After washing the cells three times with culture medium, transfer them to the culture medium according to the experimental protocol, for example, The molecules were resuspended in suction medium, mobile medium, serum-free medium, and complete medium. The enucleation efficiency was 1: 2000 dilution of Vybrant (registered trademark) Dyecycle (trademark) Green (M Olecular Probes V35004) or Hoech (1:5000 dilution) Determined in 12-well plates by adding complete MSC medium containing st 33342. A small amount of each layer was added to each well and allowed to adhere / stain in an incubator for 10 minutes. The percentage of negative cytoplasm per population was determined by reflected fluorescence microscopy.

[0231] Cytoplasmic mRNA transfection 1M cytoplasm is mixed with 1 ml of warmed amino acid-free a-MEM complete medium (Ther moFisher 12561056; 16.5% Premium Fetal Bovine Serum (FBS), 1% Glutamax (Gibco 35050061), 1% HEPES (Gibco The mRNA was suspended in 15630080). 1 μg of mRNA was diluted in heated Opti-MEM. And it was mixed with a pipette at least 20 times. 4 μl of lipofectamine-3000 (Th 46 μl of heated opti-MEM (Th) (fermoFisher L300015) Add to ermoFisher 31985062) and mix with a pipette at least 20 times. The ratio of mRNA to lipofectamine-3000 was 1:4 (w / v). Mix A and lipofectamine-3000 dilution at least 20 times using a pipette, at room temperature. The mRNA and lipofectamine-3000 mixture was incubated for 15 minutes. The cytoplasmic suspension was added, thoroughly mixed, and incubated at 37°C for 30 minutes. The cells were shaken every 5 minutes to prevent aggregation. After incubation, the cells were centrifuged. Standard a-MEM complete medium (16.5% premium FBS, 1% Antibiotic-A Resuspend in Ntimycotic (1% Glutamax, 1% HEPES) or PBS. Ta.

[0232] Cytoplasmic siRNA transfection 1M cytoplasm in 1 ml of warmed A / A-free a-MEM complete medium (16.5% p Suspended in Premium FBS (1% Glutamax, 1% HEPES). 2 μl of siRN Dilute A with warmed opti-MEM and mix at least 20 times using a pipette. 8 μl Dilute Lipofectamine-3000 with 92 μl of warmed Opti-MEM, and add a small amount. Both were mixed 20 times using a pipette. The ratio of siRNA to lipofectamine-3000 was 1:4. The result was (v / v). siRNA and lipofectamine-3000 dilution were pipetted. Mix at least 20 times and incubate at room temperature for 15 minutes. siRNA and lipof Add the Ectamin 3000 mixture to the cytoplasmic suspension, mix thoroughly, and incubate at 37°C for 20 minutes. Incubation was performed. The suspension was shaken every 5 minutes to prevent cell aggregation. 20 minutes of incubation. After incubation, the cells were centrifuged and placed in standard a-MEM complete medium (16.5% premium FBS, 1% Antibiotic-Antimycotic, 1% Glutamax, The sample was resuspended in 1% HEPES.

[0233] Generation of oncolytic virus-infected cytoplasm One day before enucleation (usually 18 hours before), 2.5*10 6 Individual hTERT-MSC The seeds were sown in a 15cm dish. Approximately 2 hours after sowing, the cells were washed once with PBS. Then, the fine In the cells, oHSV-GFP (Imanis) with different MOIs (e.g., 0.05 or 0.5) OV3001) was infected using 8 mL of serum-free opti-MEM. Next, the cells were finely divided. The cells were incubated at 37°C for 2 hours, shaking occasionally. Then the viral inoculum was... Discarded. 20 mL of pre-warmed complete culture medium (a-MEM, 16.5% premium F BS, 1% Antibiotic-Antimycotic, 1% Glutamax, 1% HEPES was added to each well. Cells were incubated at 37°C until nucleus enucleation occurred.

[0234] Lentiviruses that overexpress functional proteins in the cytoplasm Place 1-2 × 10⁶ target cells in one well of a 6-well plate. 5 In terms of cell / well density, Alternatively, seeds were sown in 10cm plates containing 0.5-1M MSC. The following day, concentrated recombinant The lentivirus was thawed in a 37°C water bath and immediately removed from the bath after thawing. Next, cells Washed three times with PBS. 200 μL of serum-free medium or 2 mL of serum-free medium (1:12 50 SureENTRY was added. Target cells were placed in a 6-well plate. The cells were infected at a 10:1 ratio. The following day, the viral supernatant was removed, and a suitable complete growth medium was added to the cells. After 72 hours of incubation, the cells were subcultured in 2 x 100 mm dishes. Appropriate amount A selective agent (i.e., puromycin) was added to generate a stable cell line. After 10-15 days, clones were selected for expansion, and positive clones were screened. The selected positive clones were enlarged for enucleation. The manipulated cytoplasm was prepared as described above. The expression of target proteins on the cytoplasm can be measured by conventional biochemical methods or functional assays, e.g. For example, fluorescence-activated cell sorting (FACS), Western blotting, or Boyden chamber. —Determined by assay.

[0235] Peptide loading into the cytoplasm 1 x 10 per well 5 / ml of complete MSC medium [MEM 1X (Gibco 1 2561-056); 16.5% Premium FBS (Atlanta Biologie) s SI 150);1%HEPES 1M(Gibco 15630-80);1%A nti-Anti 100X (Gibco 15240-062); 1% Glutamax 100X (Gibco 35050-061) 4-chamber glass slide (La Seeds were seeded on bTek II 4-chamber glass slides (155383). The cells were then... The cells were left attached for at least one hour or overnight. Then, the cells were immersed in PBS (Gibco 14190- 144) Rinse. Arg9(FAM)(10mM, Anaspec, AS-6120 7) was diluted in complete medium to a total concentration of 1:100 (100 μM). Then, the cytoplasm... The body was incubated for 1-2 hours and rinsed three times with PBS. Hoechst 33342 (Invitrogen) was added to complete medium at a 1:5000 dilution for at least 10 minutes. Next, the cells were washed with PBS and imaged using epifluorescence microscopy.

[0236] Generation of preclinical syngeneic tumor models in immunocompetent mice The fur in small sections on both sides of the mouse's flanks (from elbow level to the upper thigh, and just from the abdomen to the back) I shaved (up to the middle). I wiped off any excess hair with an alcohol wipe. 27G 1 / 2” needle Using a 1 mL tuberculin syringe equipped with a 1 M / 100 μL tuberculin syringe, inject 1 M / 100 μL E0771 cells. The injection was administered to both sides of the mussel. Continued until the tumor reached a diameter of 0.7-1.0 cm, approximately 10-20 days later. The mouse was monitored.

[0237] Intratumoral delivery of therapeutic cytoplasm The manipulated cytoplasm (i.e., loaded with IL-12 mRNA) is used as desired. The concentration was adjusted, for example, by resuspending in PBS at 3M / 50uL. On the day of injection, the animal's body weight and tumor size were measured. Dimensions were measured. Manipulated cytoplasm was injected into the center of the tumor. The mouse was monitored. Tumors and body weight were measured every 2-3 days.

[0238] Intravenous delivery of therapeutic cytoplasm The manipulated cytoplasm (i.e., loaded with IL-12 mRNA) is used as desired. The solution was resuspended in PBS at a concentration, for example, 3M / 50μL. The recommended maximum injection volume was 100μL. The injection was administered using a 1 mL tuberculin syringe and a 27G 1 / 2” or 28G 1 / 2” syringe. The procedure was performed with a needle. For intravenous (IV) injections, the Institutional Animal Care and Use Committee (IACUC) was consulted. The protocol was followed. The posterior orbital injection was administered as a ketamine / xylazine intraperitoneal (IP) injection. Alternatively, it was performed under anesthesia by isofluorane inhalation. Tail vein injection was performed using restraint devices. carried out.

[0239] [Example 6] - 3D cultured MSCs can be enucleated, and 3D-derived cytoplasm is better in vivo. It exhibits a specific in vivo distribution. MSCs are cultured in a 3D hanging drop (3D MSC), and then enucleated to form 3D cells. A substance was generated. The 3D culture protocol for MSCs using hanging drops is Curr Pro toc Stem Cell Biol. 2014 Feb 6; 28: Unit-2B.6.(Thomas J. Bartosh1 and Joni H. Yl I changed ostalo.

[0240] Healthy MSCs were harvested from 2D culture plates using trypsin, and fresh a-MEM( ThermoFisher 12561056) Complete Medium (16.5% Premium FBS) , 1% Antibiotic-Antimycotic, 1% Glutamax, 1% HE Resuspended in PES at 1.43 million cells / ml. Open the lid of the 15cm plate completely and 20 ml of PBS was added to the plate. Using a multichannel pipette, the lid of the plate was filled with PBS. Droplets of 35 μl each were prepared (approximately 50,000 cells / droplet). Approximately 100-120 One droplet was placed on each lid. The lids were closed and the plate was returned to the incubator. Two droplets were placed on each lid. The cells were cultured for several days, then harvested using a cell lifter and collected in 15 ml tubes (per tube). (Approximately 300 drops). The tube was centrifuged at 1,200 rpm for 5 minutes. Remove the supernatant. The tube was then washed twice with PBS. Next, all PBS was removed and 7.5 ml of fresh water was added. Thawed 0.25% trypsin-EDTA (ThermoFisher 25200) 114) was added to each tube. The tubes were incubated in a water bath for 4 minutes. Gently pipette approximately 10-20 times using a 1 ml pipette with a low-retention tip, then bathe in water. Then incubated for another 4 minutes. The droplets were left in a low reservoir until most of them dissociated. Using a 1ml pipette with a tip, the mixture was gently pipetted again approximately 10-20 times. 7.5ml Complete serum medium (Glutamax Supplement (Gibco 350500) 61); Fetal Bovine Serum - Premium Select (Atlanta Biologicals) SI 1550);HEPES(1M)(Gibco 15630080);Anti biotic-Antimycotic(100X)(Gibco 15240062) The solution was added to each tube, and the tubes were centrifuged at 1,200 rpm for 10 minutes. The cells were washed with 10 ml of complete serum medium and then resuspended in 5 ml of complete serum medium. The cells were passed through a 70 μm cell filter, and then the filter was washed with 5 ml of complete serum medium. The cells were counted and re-treated with 12.5% ​​Ficol at a higher concentration than 10 M / ml. The sample was cloudy. 30-40M cells were used in each nucleation tube. Subsequently, the above nucleation protocol was used. I followed the rules.

[0241] DiD-labeled conventional 2D cultured MSCs (2D MSCs), 3D MSCs, or 3D microclusters The cytoplasm was injected posteriorly into BalB / C mice. The tissues shown were obtained 24 hours after injection. Later, cells were collected and analyzed by FACS using DiD-labeled cells. Figure 24 shows the results from 3D cultured MSCs. This demonstrates the successful generation of 3D-derived cytoplasm, and shows the results of 2D culture after injection of 3D-derived cytoplasm into circulation. It was also shown that these cells have fewer lung traps than nutrient cells and exhibit good in vivo distribution to peripheral organs. This significantly improves the ability to locate cargo and deliver it to the tissue. This is what is expected.

[0242] Other Embodiments Although the present invention has been described in relation to its detailed description, it should be understood that the foregoing description is illustrative of, and not limiting, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The present invention may include the following embodiments. [1] A method comprising the step of administering a therapeutically effective amount of a composition comprising a first cytoplasm expressing or containing at least one therapeutic agent to a target. [2] The method according to [1], wherein the therapeutic agent is selected from the group consisting of therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutic agents, therapeutic gene editing factors, small molecule therapeutic agents, nanoparticles, bacteria, bacterial spores, bacteriophages, bacterial components, viruses, exosomes, lipids, and ions. [3] The method according to either [1] or [2], wherein the cells are cells taken from or derived from the subject, or derived from a cell line, immortalized cells, or cancer cells. [4] The method according to any one of [1] to [3], wherein the first cytoplasm is fused to the second cytoplasm. [5] The method according to any one of [1] to [4], further comprising the step of administering one or more additional therapies to the subject. [6] The method according to any one of [1] to [5], wherein the first cytoplasm expresses an immune system evasion region. [7] The method according to [6], wherein the immune system evasion portion is CD47. [8] The method according to any one of [1] to [7], wherein the first cytoplasm or the cell from which the first cytoplasm is obtained is manipulated to express the therapeutic agent. [9] The method according to any one of [1] to [8], wherein the first cytoplasm or the cell from which the first cytoplasm is obtained is not manipulated to express the therapeutic agent.

[10] A cytoplasm containing at least one therapeutic agent.

[11] The cytoplasm described in

[10] is selected from the group consisting of therapeutic DNA molecules, therapeutic RNA molecules, therapeutic proteins, therapeutic peptides, small molecule therapeutic agents, therapeutic gene editing factors, small molecule therapeutic agents, nanoparticles, bacteria, bacterial spores, bacteriophages, bacterial components, viruses, exosomes, lipids, and ions.

[12] The cytoplasm according to either

[10] or

[11] , further comprising an immune system evasion portion of the cytoplasm.

[13] Steps to introduce therapeutic agents into cells; The step of enucleating the aforementioned cells A method for producing cytoplasm, including [a specific component].

[14] The method according to

[13] , wherein the introduction step precedes the denucleation step.

[15] The method according to

[14] wherein the introduction step brings about the permanent expression of the therapeutic agent.

[16] The method according to

[13] , wherein the denucleation step precedes the introduction step.

[17] The method according to any one of

[13] ,

[14] , or

[16] , wherein the introduction step brings about a transient manifestation of the therapeutic agent.

[18] The method described in any one of

[13] to

[17] , wherein the introductory step includes transfection.

[19] The method according to any one of

[13] to

[17] , wherein the introduction step includes electroporation, microinjection, cell squeezing, sonoporation, impalement, or hydrodynamic delivery.

[20] A method for treating a subject, comprising the step of administering to the subject a therapeutically effective amount of a cytoplasm described in any one of

[10] to

[12] .

Claims

1. A composition for delivering a therapeutic agent to a target, The composition comprises enucleated cells derived from nucleated parent cells of mammals. The nucleated parent cells of the mammal mentioned above include stem cells or immune cells. The enucleated cells comprise (i) a therapeutic agent, and (ii) a target region comprising SDF-1α, CCL2, or a combination thereof. A composition in which, when the enucleated cells are administered systemically to a subject, the enucleated cells in the capillaries of the subject exhibit less capture of the enucleated cells in the capillaries of the subject compared to the capture of the nucleated parent cells of the mammal.

2. The composition according to claim 1, wherein systemic administration of the enucleated cells reduces the risk of the subject experiencing pulmonary embolism compared to systemic administration of nucleated cells which are identical to the enucleated cells except that they are not enucleated.

3. The composition according to claim 1, wherein when the enucleated cells are administered systemically by intranasal, subcutaneous, intra-arterial, subarachnoid, or intravenous administration, less capture is observed in the target capillaries.

4. The composition according to claim 1, wherein the therapeutic agent is used for the treatment of a disease or disorder of the target lung tissue.

5. The composition according to claim 1, wherein the therapeutic agent is used for the treatment of cancer.

6. The composition according to claim 5, wherein the cancer is metastatic cancer.

7. The composition according to claim 5, wherein the cancer is breast cancer.

8. The composition according to claim 1, wherein the therapeutic agent comprises a therapeutic small molecule, a therapeutic nucleic acid molecule, or a therapeutic peptide.

9. The composition according to claim 1, wherein the therapeutic agent comprises two or more therapeutic small molecules, therapeutic nucleic acid molecules, therapeutic peptides, or combinations thereof.

10. The composition according to claim 1, wherein the therapeutic agent comprises a cytokine or a fragment thereof.

11. The composition according to claim 10, wherein the cytokine is interleukin 12 (IL-12).

12. The composition according to claim 1, wherein the therapeutic agent comprises an inhibitor of programmed cell death ligand 1 (PD-L1).

13. The composition according to claim 12, wherein the PD-L1 inhibitor comprises a PD-L1-specific antibody or an antigen-binding fragment thereof.

14. The composition according to claim 1, wherein the therapeutic agent is expressed on the cell surface of the enucleated cells.

15. The composition according to claim 1, wherein the therapeutic agent is secreted by the enucleated cells.

16. The composition according to claim 1, which, when measured in vivo using a method comprising (i) to (iii) below, shows less capture of enucleated cells in the capillaries of the subject compared to the capture of nucleated parent cells of the mammal: (i) Labeling the enucleated cells and the nucleated parent cells using DiIC18(5); 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD), (ii) Injecting the enucleated cells and the nucleated parent cells into a living organism, (iii)Measure the in vivo distribution of the enucleated cells compared with the in vivo distribution of the nucleated parent cells using fluorescence-activated cell sorting (FACS) 24 hours after injection of (iii).

17. The composition according to claim 1, wherein the enucleated cells have a diameter of 60% of that of the nucleated parent cells of the mammal.

18. The composition according to claim 1, wherein the enucleated cells have a diameter of 5 to 20 μm.

19. The composition according to claim 1, wherein the nucleated parent cells of the mammal are not erythrocyte progenitor cells or platelets.

20. The composition according to claim 1, wherein the stem cells include mesenchymal stem cells.

21. The composition according to claim 1, wherein the immune cells include natural killer cells, macrophages, or neutrophils.

Images