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WO2025216537A1 - Composition pour l'administration de substance fonctionnelle et ses utilisations - Google Patents

Composition pour l'administration de substance fonctionnelle et ses utilisations

Info

Publication number
WO2025216537A1
WO2025216537A1 PCT/KR2025/004755 KR2025004755W WO2025216537A1 WO 2025216537 A1 WO2025216537 A1 WO 2025216537A1 KR 2025004755 W KR2025004755 W KR 2025004755W WO 2025216537 A1 WO2025216537 A1 WO 2025216537A1
Authority
WO
WIPO (PCT)
Prior art keywords
composition
extracellular
vesicular
functional
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2025/004755
Other languages
English (en)
Korean (ko)
Inventor
김기범
김은혜
김성현
김재현
이수진
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shiftbio Inc
Original Assignee
Shiftbio Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shiftbio Inc filed Critical Shiftbio Inc
Publication of WO2025216537A1 publication Critical patent/WO2025216537A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/606—Nucleosides; Nucleotides; Nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5176—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/86—Products or compounds obtained by genetic engineering

Definitions

  • the present invention relates to a composition for functional material delivery and its use.
  • lipid nanoparticles include lipid nanoparticles, viral particles, and various synthetic carriers.
  • these carriers have relatively low biocompatibility and high immunogenicity, which significantly limit their safety during repeated administration.
  • most carriers when administered systemically, most carriers accumulate in the liver and spleen, reducing drug delivery efficiency to the desired target tissues and cells.
  • mammalian cell membranes act as a biological barrier, making them largely impermeable to externally introduced pharmacologically active substances, a major obstacle to efficient drug delivery.
  • Extracellular vesicles have recently been attracting attention as an alternative to overcome these limitations.
  • EVs are spherical particles with a lipid bilayer structure naturally secreted by various cells in the body, and possess excellent characteristics such as high biocompatibility and low immunogenicity.
  • EVs are present in most body fluids, including blood and urine, and contain biologically active substances such as proteins, mRNA, and miRNA, playing a crucial role as intercellular signaling mediators.
  • EVs possess the ability to deliver payloads through fusion with the target cell membrane and endocytosis, protecting these endogenous substances from various immune components and enzymes in plasma, enabling stable delivery.
  • EVs can efficiently load a wide range of biologically active substances, including proteins, membrane receptors, and nucleic acids, demonstrating their potential as next-generation therapeutic delivery vehicles.
  • the present inventors completed the present invention by confirming that non-vesicular extracellular particles, particularly supermere, can effectively load functional substances, such as external genes, and efficiently deliver them to cells.
  • One object of the present invention is to provide a composition for delivering a functional substance comprising non-vesicular extracellular particles (NVEPs) as an active ingredient.
  • NVEPs non-vesicular extracellular particles
  • Another object of the present invention is to provide a composition for delivering a functional substance comprising a non-vesicular extracellular particle marker protein and a non-vesicular extracellular particle comprising a target functional substance.
  • Another object of the present invention is to provide a pharmaceutical composition comprising the composition.
  • Another object of the present invention is to provide a treatment method comprising a step of administering the pharmaceutical composition.
  • Another object of the present invention is to provide a cosmetic composition comprising the composition.
  • Another object of the present invention is to provide a functional substance delivery kit comprising non-vesicular extracellular particles as an active ingredient.
  • Another object of the present invention is to provide a method for delivering a functional substance using the composition for delivering a functional substance.
  • Another object of the present invention is to provide a functional substance delivery use of a composition for functional substance delivery comprising non-vesicular extracellular particles.
  • Another object of the present invention is to provide a use of non-vesicular extracellular particles for functional substance delivery.
  • a composition for functional material delivery using non-vesicular extracellular particles of the present invention can effectively load genetic material such as protein, mRNA and/or pDNA and deliver it well to cells, and thus can be utilized as a pharmaceutical composition for treating diseases or a functional cosmetic composition.
  • Figure 1 illustrates the classification system of extracellular particles and their subclassification.
  • Figure 2 shows the manufacturing process of sub-classified substances of extracellular particles.
  • Figure 3 shows the expression of protein markers of supermere, which are distinguished from exomere and small extracellular vesicles.
  • Figure 4 shows the results of treating recipient cells with extracellular particles isolated from cells transduced with eGFP, confirming that expression of eGFP protein was induced in recipient cells regardless of whether RNase pretreatment was performed.
  • Figure 5 shows the results of confirming that among the extracellular particles isolated from cells transduced with eGFP, the particles capable of inducing eGFP expression in recipient cells are small extracellular particles, not large extracellular vesicles.
  • Figure 6 shows the results confirming that among the small extracellular particles isolated from cells transduced with eGFP, the main carriers that can effectively induce eGFP expression in recipient cells are non-vesicular extracellular particles, not small extracellular vesicles.
  • Figure 7 shows the results confirming that, regardless of the type of transduced plasmid, non-vesicular extracellular particles are the main carriers that can more effectively induce eGFP expression in recipient cells compared to small extracellular vesicles and the entire extracellular particle group.
  • Figure 8 shows the results confirming that supermere among non-vesicular extracellular particles is the main carrier that can induce eGFP expression in recipient cells more effectively than small extracellular vesicles and exomeres, regardless of the type of transduced plasmid.
  • Figure 9 shows the results of confirming the content of endogenous eGFP-mRNA in small extracellular vesicles, exomeres, and supermeres isolated from cells transduced with eGFP.
  • Figure 10 shows the results of confirming the level of endogenous eGFP protein expression in small extracellular vesicles, exomeres, and supermeres isolated from cells transduced with eGFP.
  • Figure 11 shows the results demonstrating that supermeres isolated from eGFP-transduced cells are the main delivery vehicles that can more effectively deliver eGFP mRNA into recipient cells than small extracellular vesicles and exomeres.
  • Figure 12 shows the results demonstrating that supermeres isolated from eGFP-transduced cells are the main delivery vehicles that induce eGFP protein expression in recipient cells more effectively than small extracellular vesicles and exomeres.
  • Figure 13 shows the results of confirming the internalization of supermere into cells through lipid rafts ( Figures 13a and 13b: Cy5.5 expression results in recipient cells 30 minutes, 3 hours, and 24 hours after treating recipient cells with each drug and supermere, Figure 13c: Changes in the proportion of cells containing supermere stained with Cy5.5 for 24 hours after treating recipient cells with each drug and supermere).
  • Figure 14 shows the results confirming that supermere can deliver exogenous eGFP mRNA to recipient cells more effectively than small extracellular vesicles and exomeres.
  • Figure 15 shows the results of confirming intracellular delivery of GFP pDNA using Supermere.
  • Figure 16 shows the results of ex vivo imaging of GFP fluorescence expressed in intestinal tissue to compare the intestinal exogenous eGFP mRNA delivery and protein translation efficiency of supermere and lipid nanoparticles after intravascular injection.
  • Figure 17 shows the results of confirming eGFP protein expression in intestinal tissue to compare the intestinal exogenous eGFP mRNA delivery and protein translation efficiency of supermere and lipid nanoparticles after intravascular injection.
  • Figure 18 shows the results of confirming the distribution in each organ after a certain period of time after intravascular injection of supermembrane and small extracellular vesicles derived from HEK293FT cells labeled with fluorescent dye.
  • Figure 19 is an image comparing the accumulation of supermere and small extracellular vesicles derived from HEK293FT cells in each organ by isolating only liver, spleen, and kidney tissues in the experiment of Figure 18.
  • Figure 20 shows the results of confirming the distribution in each organ after a certain period of time after intravascular injection of supermembrane and small extracellular vesicles derived from human bone marrow-derived mesenchymal stem cells (MSCs) labeled with fluorescent dye.
  • MSCs human bone marrow-derived mesenchymal stem cells
  • Figure 21 shows the results of confirming the expression level of a functional substance in a supermere isolated from cells transduced in a form in which the supermere marker protein TGFBI and the target functional substance are fused.
  • the present invention is based on the identification of a novel use for non-vesicular extracellular particles (NVEPs).
  • NVEPs non-vesicular extracellular particles
  • extracellular vesicles Conventionally, cells release vesicles of various membrane types depending on the extracellular environment. These released vesicles are usually referred to as extracellular vesicles (EV). These extracellular vesicles enable the exchange of materials such as proteins, lipids, and genetic materials between cells, and act as mediators that transmit physiological/pathological signals, and various studies are being conducted using them.
  • EV extracellular vesicles
  • the present invention is based on the novel demonstration that non-vesicular extracellular particles, particularly supermelas, can effectively load functional substances, such as external genes, and efficiently deliver them to cells.
  • one aspect according to one embodiment of the present invention provides a composition for functional material delivery comprising a non-vesicular extracellular particle (NVEP) as an active ingredient.
  • NVEP non-vesicular extracellular particle
  • the above extracellular particles are classified into vesicular and non-vesicular, and vesicles include extracellular vesicles (EV).
  • the extracellular vesicles are natural nanoparticles composed of all lipid bilayers derived from cells, and are distinguished from the non-vesicular extracellular particles (NVEPs) used as the active ingredient in the present invention.
  • NVEPs non-vesicular extracellular particles
  • the above extracellular vesicles are divided into large extracellular vesicles (large EVs) with an average diameter of 200 nm or more and small extracellular vesicles (small EVs) with an average diameter of 200 nm or less.
  • Non-Vesicular Extracellular Particle refers to a natural nanoparticle derived from a cell and without a lipid bilayer.
  • exomeres which are natural nanoparticles without a lipid bilayer of 50 nm or less
  • supermeres Supernatant of Exomere
  • the above supermere has characteristics that distinguish it from extracellular vesicles or exomeres in terms of size, RNA and protein profiles, and total RNA quantity.
  • the supermere can be distinguished from extracellular vesicles or exomeres using a marker.
  • the marker is a protein that exists specifically in large numbers in the supermere and can be named a “supermere marker protein.”
  • the supermere marker protein may be at least one selected from Transforming growth factor, beta-induced (TGFBI), Enolase-1 (ENO1), Enolase-2 (ENO2), Heat shock 70 kDa protein 13 (HSPA13), GPI (Glucose-6-phosphate isomerase), LDHA (Lactate Dehydrogenase A), TPI1 (Triosephosphate Isomerase 1), HK1 (Hexokinase 1), MDH1 (Malate Dehydrogenase 1), NUCB1 (Nucleobindin-1), and PDIA4 (Protein Disulfide Isomerase Family A Member 4), but is not limited thereto.
  • the supermembrane can be distinguished by the marker regardless of the type of derived cell.
  • the above cells may be, for example, human embryonic kidney cells (HEK cells) or stem cells, but are not limited thereto.
  • HEK cells human embryonic kidney cells
  • stem cells but are not limited thereto.
  • the stem cells may be embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or adult stem cells.
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • adult stem cells adult stem cells.
  • the adult stem cells may be selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, multipotent stem cells, and amniotic epithelial cells.
  • the mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta.
  • the non-vesicular extracellular particle may be, but is not limited to, a supermere.
  • the functional material may be in the form of a protein.
  • the functional material may be in the form of RNA.
  • the functional material may be in the form of DNA.
  • the functional material may be, for example, a gene, and may be selected from the group consisting of, but not limited to, mRNA, shRNA, miRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, IncRNA, ribozyme, mini-circle DNA, or plasmid DNA (pDNA) as a specific example.
  • the above genes may exist in nature or be synthesized, and may exist in various sizes, from oligonucleotides to chromosomes. These genes may originate from, but are not limited to, humans, animals, plants, bacteria, viruses, etc.
  • the gene may be a newly synthesized synthetic gene, and may be a gene synthesized for various purposes, such as a gene as a drug, a gene as an inhibitor, or a gene having a cosmetic effect, and may be delivered by being loaded onto the non-vesicular extracellular particle of the present invention without limitation.
  • the functional material may include a protein.
  • the protein may be, but is not limited to, an antibody or an immunologically active fragment thereof, an intrabody, a single chain variable fragment, an affibody, an enzyme, a transporter, a tumor suppressor, a virus or bacterial inhibitor, a cellular component protein, a DNA or RNA binding protein, a DNA repair inhibitor, a nuclease, a proteinase, an integrase, a transcription factor, a growth factor, an apoptosis inhibitor or inducer, a toxin, a structural protein, a neurotrophic factor, a membrane transporter, a nucleotide binding protein, a heat shock protein, or a CRISPR-associated protein.
  • an antibody or an immunologically active fragment thereof an intrabody, a single chain variable fragment, an affibody, an enzyme, a transporter, a tumor suppressor, a virus or bacterial inhibitor, a cellular component protein, a DNA or RNA binding protein, a DNA repair inhibitor, a nucle
  • the functional material may include a drug.
  • Such a drug may be any small molecule compound drug such as a cytotoxic anticancer agent, any biopharmaceutical such as a recombinant protein, siRNA, ASO, mRNA, gRNA, tRNA, etc., and in terms of efficacy, it may be an anti-inflammatory agent, analgesic, anti-arthritic agent, antispasmodic agent, antidepressant agent, antipsychotic agent, tranquilizer, anti-anxiety agent, narcotic antagonist, anti-Parkinson's disease drug, cholinergic agonist, anticancer agent, angiogenesis inhibitor, immunosuppressant, immunostimulant, antiviral agent, antibiotic, appetite suppressant, anticholinergic agent, antihistamine, antimigraine agent, hormonal agent, coronary vascular agent, vasodilator, contraceptive, antithrombotic agent, diuretic, antihypertensive agent, cardiovascular
  • a drug may be any small molecule compound drug such as a cytotoxic anticancer agent
  • the functional substance may be a therapeutic agent, a diagnostic agent, or a combination thereof.
  • the above functional material may be selected from the group consisting of nucleic acids, proteins, polypeptides, low molecular weight compounds and carbohydrates.
  • the functional material may comprise one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and/or polypeptides, one or more cellular organelles, or any combination thereof.
  • the functional material may comprise one or more cellular components.
  • the functional material may comprise one or more cytoplasmic and/or nuclear components.
  • the functional material may be a nucleic acid, for example, a transcription factor, DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-coding DNA, a gene, an operon, a chromosome, a genome, a transposon, a retrotransposon, a viral genome, an intron, an exon, a modified DNA, ssDNA (single-stranded DNA), dsDNA (double-stranded DNA), mRNA (messenger RNA), sgRNA (single guide RNA), gRNA (guide RNA), pegRNA (prime editing guide RNA), tRNA (transfer RNA), a modified RNA, microRNA (miRNA), siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA),
  • the nucleic acid is a wild-type nucleic acid. In some embodiments, the nucleic acid is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of a plurality of nucleic acid sequences.
  • the nucleic acid may contain a nuclear localization signal (NLS) that can enhance editing efficiency within the nucleus.
  • NLS nuclear localization signal
  • An NLS is an amino acid sequence that tags a protein for nuclear transport into the cell nucleus.
  • the functional material may comprise a nucleic acid.
  • the target material may comprise RNA that enhances the expression of an endogenous protein (e.g., endogenous to the cell producing the lipid bilayer particle, and endogenous to the target cell, in some embodiments), or siRNA or miRNA that inhibits protein expression of an endogenous protein.
  • the functional material is a polypeptide, for example, an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motor polypeptide, a defense polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein binding polypeptide, a lipid binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, an apoptotic polypeptide, a nuclear transport polypeptide, a nucleic acid binding polypeptide, a reprogramming
  • the functional agent comprises a small molecule, such as an ion (e.g., Ca 2+ , Cl - , Fe 2+ ), a carbohydrate, a lipid, a reactive oxygen species, a reactive nitrogen species, an isoprenoid, a signaling molecule, a heme, a polypeptide cofactor, an electron accepting compound, an electron donating compound, a metabolite, a ligand, and any combination thereof.
  • an ion e.g., Ca 2+ , Cl - , Fe 2+
  • a carbohydrate e.g., a carbohydrate, a lipid, a reactive oxygen species, a reactive nitrogen species, an isoprenoid, a signaling molecule, a heme, a polypeptide cofactor, an electron accepting compound, an electron donating compound, a metabolite, a ligand, and any combination thereof.
  • the functional agent comprises a mixture of proteins, nucleic acids, or metabolites, e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any combination thereof.
  • proteins, nucleic acids, or metabolites e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); multiple regulatory RNAs; and any
  • the functional material comprises one or more organelles, such as chondrosomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasms, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, myofibrils, nematodes, peroxisomes, proteasomes, vesicles, stress granules, networks of organelles, and any combination thereof.
  • organelles such as chondrosomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasms, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosome
  • the functional agent may be a DNA digesting agent capable of digesting DNA. This refers to an agent capable of cleaving bonds between nucleotide subunits of a nucleic acid (e.g., phosphodiester bonds).
  • the DNA digesting agent is a nuclease.
  • a nuclease is an enzyme that hydrolyzes nucleic acids.
  • Nucleases can be classified as endonucleases or exonucleases. Endonucleases are a group of enzymes that catalyze the hydrolysis of bonds between nucleic acids within a DNA or RNA molecule. Exonucleases are a group of enzymes that catalyze the hydrolysis of single nucleotides at the ends of DNA or RNA chains. Nucleases can also be classified based on whether they specifically digest DNA or RNA.
  • Nucleases that specifically catalyze the hydrolysis of DNA can be referred to as deoxyribonucleases, or DNases, while nucleases that specifically catalyze the hydrolysis of RNA can be referred to as ribonucleases, or RNases.
  • Some nucleases are specific for single-stranded or double-stranded nucleic acid sequences.
  • Some enzymes have both exonuclease and endonuclease properties. Additionally, some enzymes can digest both DNA and RNA sequences.
  • the functional agent may be an endonuclease.
  • endonucleases include, but are not limited to, zinc finger nucleases (ZFNs), ZFN dimers, ZFNickases, transcription activator-like effector nucleases (TALENs), meganucleases, or RNA-guided DNA endonucleases (e.g., the CRISPR/Cas system).
  • the endonuclease may be engineered, chimerized, or isolated from an organism.
  • the endonuclease may be engineered to recognize a specific DNA sequence, for example, by mutagenesis.
  • the functional agent may be an RNA-guided DNA endonuclease (e.g., a CRISPR-Cas system).
  • the lipid bilayer particle may additionally include gRNA, crRNA, tracrRNA, etc.
  • gRNA RNA-guided DNA endonuclease
  • guide RNA CRISPR guide sequence
  • the terms "gRNA,” “guide RNA,” and “CRISPR guide sequence” may be used interchangeably and refer to a nucleic acid comprising a sequence that determines the specificity of the Cas DNA binding protein of the CRISPR/Cas system.
  • the gRNA hybridizes (partially or fully complementarily) to a target nucleic acid sequence in the host cell genome.
  • the gRNA or portion thereof that hybridizes to the target nucleic acid may be 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length.
  • the length of the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides.
  • the gRNA sequence does not include a scaffold sequence, and the scaffold sequence is expressed as a separate transcript.
  • the gRNA sequence further includes an additional sequence that is complementary to a portion of the scaffold sequence and functions to bind (hybridize) the scaffold sequence and recruit an endonuclease to the target nucleic acid.
  • the RNA-guided DNA endonuclease is a Cas enzyme, i.e., a CRISPR-associated endonuclease.
  • the Cas enzyme can be a naturally occurring Cas enzyme or a functional derivative thereof. In certain embodiments, the Cas enzyme can comprise one or more mutations.
  • the Cas enzyme can be a type II, type I, type III, type IV, or type V CRISPR system enzyme.
  • the Cas enzyme is a Cas9 enzyme (also known as Cas5, Csn1, or Csx12). Cas9 can be wild-type or mutant.
  • the endonuclease is a Cas9 homolog or ortholog.
  • Cas9 may be any variant disclosed in U.S. Patent Publication No. US 2014/0068797 A1, which is incorporated herein by reference.
  • the Cas9 enzyme can be type II-A, type II-B or type II-C.
  • the Cas9 enzyme may be derived from various species.
  • Non-limiting examples of Cas9 enzymes may include Cas9 derived from Streptococcus pyogenes ( S. pyogenes ), Streptococcus pneumoniae ( S. pneumoniae ), Staphylococcus aureus , Neisseria meningitidis , Streptococcus thermophilus ( S. thermophilus ), or Treponema denticola .
  • the Cas9 enzyme may also be derived from a microorganism of the genus Corynebacter , Sutterella , Legionella , Treponema , Filifactor , Eubacterium , Streptococcus , Lactobacillus , Mycoplasma , Bacteroides , Flaviivola , Flavobacterium , Sphaerochaeta , Azospirillum , Gluconacetobacter , Neisseria , Roseburia , Parvibaculum , Staphylococcus , Nitratifractor , Mycoplasma , or campylobacter .
  • the Cas enzyme is Cas9, Cpf1, C2c1, C2c2, C2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Cs f1, Csf2, Csf3, Csf4, It may be a homologue, an ortholog thereof, or a variant thereof.
  • the functional material may comprise a diagnostic agent, including but not limited to a radiotracer or radionuclide used in positron emission tomography (PET) (e.g., carbon-11, nitrogen-13, oxygen-15, and fluorine-18).
  • PET positron emission tomography
  • these agents can be more precisely delivered to the tissue of interest by loading them into engineered extracellular particles that target the tissue of interest, i.e., the tissue being scanned, either artificially or naturally, thereby reducing off-target delivery of the diagnostic agent.
  • the functional material may be a targeting moiety.
  • the non-vesicular extracellular particle may additionally comprise at least one targeting moiety.
  • the targeting moiety can be used to target the non-vesicular extracellular particle to a specific organ, tissue, or cell for delivery of the functional agent using the non-vesicular extracellular particle.
  • the targeting moiety can bind to a marker (or target molecule) expressed in a cell or cell population.
  • the marker may be expressed on various cell types, for example, all antigen-presenting cells (e.g., dendritic cells, macrophages, and B lymphocytes).
  • all antigen-presenting cells e.g., dendritic cells, macrophages, and B lymphocytes.
  • the marker may be expressed only in a particular cell population (e.g., dendritic cells).
  • markers expressed in a particular cell population include C-type lectin domain family 9 member A (CLEC9A) protein or dendritic cell-specific intercellular adhesion molecule-3-grafted non-binding protein: integrin (DC-SIGN), CD207, CD40, Clec6, dendritic cell immunoreceptor (DCIR), DEC-205, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), MARCO, Clec12a, DC-asialoglycoprotein receptor (DC-ASGPR), DC immunoreceptor 2 (DCIR2), Dectin-1, macrophage mannose receptor (MMR), BDCA-1 (CD303, Clec4c), Dectin-2, Bst-2 (CD317), and any combination thereof.
  • DC-SIGN C-type lectin domain family 9 member A
  • DCIR dendritic cell-specific intercellular
  • the targeting moiety can be an antibody or an antigen-binding fragment thereof.
  • Antibodies and antigen-binding fragments thereof include whole antibodies, polyclonal, monoclonal, and recombinant antibodies, and fragments thereof, and may further include single chain antibodies, humanized antibodies, murine antibodies, chimeric antibodies, mouse-human, mouse-primate, and primate-human antibodies. Monoclonal antibodies, anti-idiotypic antibodies, antibody fragments (e.g., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments), diabodies, and antibody-related polypeptides.
  • Antibodies and antigen-binding fragments thereof can include bispecific antibodies and multispecific antibodies, as long as they exhibit the desired biological activity or function.
  • the supermere may have improved functional substance delivery capacity compared to extracellular vesicles or exomeres.
  • the supermere may accumulate in a smaller amount in the liver compared to extracellular vesicles or exomeres.
  • the supermere may accumulate in a smaller amount in the spleen compared to extracellular vesicles or exomeres.
  • the supermere may accumulate in a larger amount in the kidney compared to extracellular vesicles or exomeres.
  • the composition may comprise a vector comprising the gene.
  • vector refers to a carrier into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence.
  • the nucleic acid sequence may be exogenous or heterologous.
  • examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages).
  • the vector includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector.
  • Suitable vectors include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and can be manufactured in various ways depending on the purpose.
  • the promoter of the vector may be constitutive or inducible.
  • the vector may be, but is not limited to, pcDNA3.1, pcDNA3.4, pMXs-IRES, pDisplay, pcDNa3.4, pRG2-GG, prp[exp] or pCMV.
  • composition for functional substance delivery comprising a non-vesicular extracellular particle comprising a non-vesicular extracellular particle marker protein and a target functional substance.
  • the target functional material may be in a form fused with a non-vesicular extracellular particle marker protein.
  • the non-vesicular extracellular particles may be supermeers.
  • the target functional material may be in a form fused with a supermere marker protein.
  • the supermembrane marker protein may be at least one selected from the group consisting of Transforming growth factor, beta-induced (TGFBI), Enolase-1 (ENO1), Enolase-2 (ENO2), Heat shock 70 kDa protein 13 (HSPA13), Glucose-6-phosphate isomerase (GPI), Lactate Dehydrogenase A (LDHA), Triosephosphate Isomerase 1 (TPI1), Hexokinase 1 (HK1), Malate Dehydrogenase 1 (MDH1), Nucleobindin-1 (NUCB1), Protein Disulfide Isomerase Family A Member 4 (PDIA4), fragments thereof, mutants thereof, mutants of fragments thereof, and fragments of mutants thereof, but is not limited thereto.
  • TGFBI beta-induced
  • ENO1 Enolase-1
  • ENO2 Enolase-2
  • HSPA13 Heat shock 70 kDa protein 13
  • GPI Glucose-6-phosphate isomerase
  • LDHA
  • compositions for delivering the functional substance comprising the composition for delivering the functional substance.
  • the pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.
  • Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion, or into a pill, capsule, granule, or tablet.
  • the pharmaceutical composition of the present invention may be a patch, a liquid, a pill, a capsule, a granule, a tablet, a suppository, or the like.
  • These preparations can be prepared by conventional methods used in formulation in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be formulated into various preparations depending on each disease or ingredient.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not causing side effects.
  • the effective dosage level may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field.
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses.
  • the daily dosage of the compound of chemical formula 1 of the present invention is about 0.01 to 1000 mg/kg, preferably 0.1 to 100 mg/kg, and can be administered once or several times a day.
  • administration means introducing a predetermined substance into a patient by an appropriate method, and the administration route of the composition may be administered through any common route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention may be administered by any device that allows the active substance to move to the target tissue.
  • it may be administered by transdermal administration, oral administration, intrathecal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, inner ear administration, intrauterine epidural administration, sublingual administration, and intracerebrovascular injection, but is not limited thereto.
  • the pharmaceutical composition may appropriately contain, if necessary, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc. depending on the administration method or formulation.
  • a suspending agent e.g., a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc.
  • the pharmaceutical composition may be manufactured in a unit dose form or may be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains.
  • the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition.
  • excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc may be used.
  • Oral liquid preparations include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • Suppository bases include withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.
  • injections can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.
  • the route of administration of the pharmaceutical composition of the present invention may be through any general route as long as it can reach the target tissue, but may be through subcutaneous injection using an osmotic pump, intradermal injection, intravenous injection, intraperitoneal injection, intravitreal injection, oral administration, etc.
  • prevention used in the present invention means any act of suppressing a disease or delaying its onset by administering the composition.
  • treatment means any act in which the symptoms of a disease are improved or beneficially changed by administering the composition.
  • subject of the present invention refers to any animal that has developed or may develop a disease, and typically may be an animal that can show a beneficial effect by treatment using the composition of the present invention, but includes without limitation any subject that has symptoms of the disease or is likely to have such symptoms.
  • the pharmaceutical composition of the present invention can be administered as an individual therapeutic agent, or can be administered in combination with existing disease therapeutic agents, and can be administered sequentially or simultaneously with the existing therapeutic agents.
  • another aspect according to one embodiment of the present invention provides a treatment method comprising a step of administering the pharmaceutical composition.
  • another aspect according to one embodiment of the present invention provides a cosmetic composition comprising the composition for delivering the functional material.
  • the composition can be formulated in various forms by adding various components as auxiliary components for delivery and stabilization, etc.
  • the cosmetic composition may have a formulation such as a mist, serum, nourishing toner, emulsifying toner, emulsion, suspension, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, powder, makeup base, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, facial cleanser, treatment, beauty liquid, beauty pack, ointment, gel, liniment, liquid, patch, spray, bath agent, sunscreen, sun oil, and hair product.
  • the scope of the formulation is not limited thereto, and the formulation of the cosmetic composition may be manufactured into any formulation commonly manufactured in the art.
  • the cosmetic composition may further comprise a cosmetically acceptable carrier.
  • the type of the cosmetically acceptable carrier of the present invention is not particularly limited, as long as it does not inhibit the activity and properties of the cosmetic composition of the present invention, and any cosmetically acceptable carrier commonly used in the art may be used.
  • Non-limiting examples of the cosmetically acceptable carrier include saline solution, sterile water, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, and ethanol. These may be used alone or in combination of two or more.
  • the cosmetically acceptable carrier varies depending on the formulation of the cosmetic composition.
  • the above formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.
  • lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, when it is a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether.
  • a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
  • a liquid diluent such as water, ethanol or propylene glycol
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.
  • alkali metal salts of fatty acids fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto.
  • the formulation of the above cosmetic composition is a pack, it includes all forms of a peel-off pack containing polyvinyl alcohol or the like, a wash-off pack containing pigments such as kaolin, talc, zinc oxide, or titanium dioxide in a general emulsified cosmetic, or a mask sheet pack, but is not particularly limited thereto.
  • the ingredients included in the above cosmetic composition may include, in addition to the composition for delivering functional substances as an active ingredient, ingredients commonly used in cosmetic compositions, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, preservatives, moisturizers, pigments, bactericides, antioxidants, surfactants, vitamins, pigments, and fragrances.
  • the above cosmetic composition may additionally include a skin absorption promoter to enhance its effectiveness.
  • the composition may further include one or more active ingredients exhibiting the same or similar efficacy.
  • the composition for delivering the functional material is present in an amount of 0.000000001 wt% to 90 wt%, for example, 0.000000001 wt% to 80 wt%, 0.000000001 wt% to 60 wt%, 0.000000001 wt% to 30 wt%, 0.000000001 wt% to 20 wt%, 0.000000001 wt% to 10 wt%, 0.000000001 wt% to 5 wt%, 0.000000001 wt% to 0.001 wt%, 0.000000001 wt% to 0.0001 wt%, 0.000000001 wt% to 0.00001 wt%, 0.0001 wt% to 90 wt%, 0.0001 wt% to 80 wt%, 0.0001 wt% to 60 wt%, 0.0001 wt% to 30 wt%, 0.0001 wt% to 20 wt%, 0.0001 wt%
  • the cosmetic composition comprises a functional material delivery composition having 10 5 pieces/ml to 10 10 pieces/ml, for example, 10 5 pieces/ml to 10 9 pieces/ml, 10 5 pieces/ml to 10 8 pieces/ml, 10 5 pieces/ml to 10 7 pieces/ml, 10 5 pieces/ml to 10 6 pieces/ml, 10 6 pieces/ml to 10 10 pieces/ml, 10 6 pieces/ml to 10 9 pieces/ml, 10 6 pieces/ml to 10 8 pieces/ml, 10 6 pieces/ml to 10 7 pieces/ml, 10 7 pieces/ml to 10 10 pieces/ml, 10 7 pieces/ml to 10 9 pieces/ml, 10 7 pieces/ml to 10 8 pieces/ml, 10 It may be included in a concentration of 8 /ml to 10 /ml, 10 /ml to 10 /ml, or 10 /ml to 10 /ml.
  • Another aspect according to one embodiment of the present invention provides a method for preparing a composition for functional material delivery, comprising the following steps.
  • the centrifugation in step (c) above can be performed initially at 5,000 g to 15,000 g, thereby obtaining large extracellular vesicles or small extracellular particles.
  • a second centrifugation can be performed at 100,000 g to 200,000 g, through which small extracellular vesicles or non-vesicular extracellular particles can be obtained.
  • a tertiary centrifugation can be performed at 150,000 g to 200,000 g, through which exomeres can be obtained.
  • a fourth centrifugation can be performed at 350,000 g to 400,000 g, through which a supermeer can be obtained.
  • the cell of step (a) may be one into which a functional material, for example a gene, has been introduced, but is not limited thereto.
  • the manufacturing method may further include a step of mixing the obtained supermembrane with a functional material to be delivered, for example, a gene.
  • another aspect according to one embodiment of the present invention provides a method for delivering a functional material to a cell by a non-vesicular extracellular particle (NVEP).
  • NVEP non-vesicular extracellular particle
  • kits for delivering a functional substance comprising a non-vesicular extracellular particle (NVEP) as an active ingredient.
  • NVEP non-vesicular extracellular particle
  • the carrier means is suitable for containing one or more containers, such as bottles or tubes, each container containing independent components used in the method of the present invention.
  • the kit may include a user guide.
  • a user guide is a printed document that explains how to use the kit, such as the reaction conditions provided. This includes instructions in the form of pamphlets or leaflets, labels attached to the kit, and descriptions on the packaging containing the kit.
  • the user guide includes information disclosed or provided through electronic media, such as the Internet.
  • kits for delivering a functional substance comprising a non-vesicular extracellular particle (NVEP) marker protein; and a non-vesicular extracellular particle (NVEP) comprising a target functional substance.
  • NVEP non-vesicular extracellular particle
  • NVEP non-vesicular extracellular particle
  • the above non-vesicular extracellular particle marker protein may be, but is not limited to, a non-specific protein.
  • the non-specific protein may be a protein present in a non-vesicular extracellular particle, or may be a protein present on the outside of a non-vesicular extracellular particle that moves into a non-vesicular extracellular particle when overexpressed in a cell, but is not limited thereto.
  • non-vesicular extracellular particles non-vesicular extracellular particle marker proteins, target functional substances, functional substance delivery and kits are as described above.
  • composition for functional substance delivery comprising a non-vesicular extracellular particle marker protein and a target functional substance.
  • non-vesicular extracellular particles non-vesicular extracellular particle marker protein, target functional substance and functional substance delivery are as described above.
  • another aspect according to one embodiment of the present invention provides a method for delivering a functional substance using the composition for delivering a functional substance.
  • another aspect according to one embodiment of the present invention provides a use of a functional substance delivery composition comprising non-vesicular extracellular particles for functional substance delivery.
  • another aspect according to one embodiment of the present invention provides a use of non-vesicular extracellular particles for functional material delivery.
  • Example 1 Collection method of extracellular particle sub-classes
  • Extracellular particles including supermeres, exomeres, and small extracellular vesicles, were isolated and obtained from HEK293FT cell lines and human bone marrow-derived stem cells (Fig. 2). Specifically, donor cells were seeded at 4.5 x 10 6 cells in a 150 ⁇ culture dish, and 20 ml of cell culture medium (DMEM high glucose + 10% FBS + 1% AA) was added and cultured for 48 hours. Afterwards, the supernatant was removed, washed once with DPBS, and replaced with 19 ml of serum-free medium (DMEM high glucose + 1% AA + 1% Glutamax) and pre-incubated for 30 minutes.
  • DMEM high glucose + 10% FBS + 1% AA fetal bovine serum
  • transfection reagent was prepared by mixing pcDNA3.1 and PEI in serum-free medium, incubating at room temperature for 15 minutes, and then adding the reagent to the donor cells. The medium was replaced with serum-free medium 6 hours after transduction, and the final supernatant was collected 48 hours later. Collection of extracellular particles obtained without transduction was also performed in the same manner.
  • the obtained supernatant was centrifuged at 3000 g for 5 minutes and filtered through a 0.45 ⁇ m filter (Sartolab RF500 PES). Subsequently, it was concentrated using Amicon (10 kDa filter, UFC901024, Merck) or TFF (100 kDa, S04-E100-05-N, Repligen). The supernatant was then centrifuged at 10,000 g for 30 minutes and then ultracentrifuged at 150,000 g for 90 minutes at 4 °C (Ultracentrifuge, Beckman). Small extracellular vesicles were isolated using 1X PBS-PIC (kept on ice for at least 20 min, then centrifuged at 13,000 g for 20 min).
  • the supernatant of small extracellular vesicles was transferred to a UC tube (355655) and centrifuged at 167,000 g for 16 h.
  • Exomeres were isolated using 1X PBS-PIC (kept on ice for at least 20 min, then centrifuged at 13,000 g for 20 min).
  • the exomere supernatant was transferred to a 70Ti rotor high speed tube (355618) and ultracentrifuged at 367,000 g for 16 h.
  • Supermeres were isolated from the pellet generated after ultracentrifugation using 1X PBS-PIC (kept on ice for at least 20 min, then briefly centrifuged for 2 min).
  • TGFBI 10188-1-AP-20UL
  • Enolase-1 3810T
  • Enolase-2 8071S
  • HSPA13 sc-398297
  • CD81 EXOAB-CD81A1
  • HEK293FT-derived supermere exhibited a protein expression pattern distinct from exomere and small extracellular vesicles (Fig. 3).
  • HEK293FT cells were treated with a transfection reagent (1 ml) containing pcDNA3.1-eGFP-MS2X2 (60 ⁇ g) and PEI (240 ⁇ g), and the medium was replaced with serum-free medium after 6 h, and the supernatant was collected after 48 h.
  • the plasmid used was constructed by cloning a polynucleotide (SEQ ID NO: 2) encoding the eGFP-MS2X2 protein (SEQ ID NO: 1) into the pcDNA3.1 vector (ThermoFisher).
  • HEK293FT cells which are recipient cells, were seeded in a 24-well plate at 2.8 X 10 5 cells/well (500 ⁇ l of cell culture medium). After 24 hours, the supernatant was aspirated and washed once with DPBS. The extracellular particles were well mixed in the serum-free medium and incubated for 48 hours in a final volume of 500 ⁇ l.
  • a group treated with RNase A was additionally prepared separately in the same process. The RNase A-treated group was used by incubating the extracellular particles at 37 °C for 30 minutes and then treating them with RNase A at 1 ⁇ g/ml. After 48 hours, the attached recipient cells were collected with a cell scraper (90020) and automated Western blotting (ProteinSimple) was performed.
  • Donor cells were treated with 1 ml of a transfection reagent containing 60 ⁇ g of pcDNA3.1-eGFP-MS2X2 and 240 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was collected 48 hours later.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the supernatant after 10,000 g centrifugation, and large extracellular vesicles were separated and obtained from the pellet after 18,000 g centrifugation.
  • HEK293FT cells which are recipient cells, were seeded in a 24-well plate at 2.8 ⁇ 10 5 cells/well (500 ⁇ l of cell culture medium), and the supernatant was aspirated after 24 hours and washed once with DPBS. Afterwards, the final 500 ⁇ l obtained by mixing extracellular particles, small extracellular particles, and large extracellular vesicles in serum-free medium was incubated for 24 hours. After incubation, attached recipient cells were collected with a cell scraper (90020) and automated Western blotting (ProteinSimple) was performed.
  • Example 5 Confirmation of cell delivery of functional materials loaded inside non-vesicular extracellular particles (NVEPs).
  • NVEPs non-vesicular extracellular particles
  • Donor cells were treated with 1 ml of transfection reagent containing 60 ⁇ g of pcDNA3.1-eGFP-MS2X2 + 240 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was collected 48 hours later.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the 10,000 g centrifugation supernatant, large extracellular vesicles were separated and obtained from the 18,000 g centrifugation pellet, and non-vesicular extracellular particles and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation supernatant and pellet, respectively.
  • HEK293FT cells which are recipient cells, were seeded in a 24-well plate at 2.8 X 10 5 cells/well (500 ⁇ l of cell culture medium), and the supernatant was aspirated after 24 hours and washed once with DPBS. Afterwards, the final 500 ⁇ l obtained by mixing extracellular particles, large extracellular vesicles, small extracellular particles, small extracellular vesicles, and non-vesicular extracellular particles in serum-free medium was incubated for 48 hours. After incubation, attached recipient cells were collected with a cell scraper (90020) and automated Western blotting (JESS Protein Simple) was performed.
  • JESS Protein Simple Western blotting
  • eGFP plasmid without MS2X2 was transfected under the same conditions as pcDNA3.1-eGFP-MS2X2, and then extracellular particles such as non-vesicular extracellular particles and small extracellular vesicles were separated and treated in recipient cells.
  • pcDNA3.1-eGFP plasmid a polynucleotide (SEQ ID NO: 4) encoding eGFP protein (SEQ ID NO: 3) was cloned into the pcDNA3.1 vector and used.
  • the overexpressed functional substances are mainly loaded into non-vesicular extracellular particles of less than 50 nm in size and are effectively delivered to recipient cells.
  • Example 6 Confirmation of cell delivery of functional materials loaded inside the supermembrane.
  • HEK293FT cells were used as donor and recipient cells.
  • Donor cells were treated with 1 ml of a transfection reagent containing 30 ⁇ g of pcDNA3.1-eGFP-MS2X2 or pcDNA3.1-myc MCP + 120 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained after 48 hours.
  • the pcDNA3.1-eGFP-MS2X2 plasmid was the same as that prepared in Example 2, and a polynucleotide encoding the myc-MCP protein (SEQ ID NO: 5) was cloned into the pcDNA3.1 vector to prepare the pcDNA3.1-myc MCP plasmid.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the 10,000 g centrifugation supernatant, large extracellular vesicles from the 18,000 g centrifugation pellet, non-vesicular extracellular particles and small extracellular vesicles from the 150,000 g ultracentrifugation supernatant and pellet, respectively, and supermeres and exomeres from the 167,000 g ultracentrifugation supernatant and pellet, respectively.
  • HEK293FT cells which are recipient cells, were seeded in a 24-well plate at 2.8 ⁇ 10 5 cells/well (500 ⁇ l of cell culture medium), and after 24 hours, the supernatant was aspirated and washed once with DPBS. Afterwards, each nanoparticle was well mixed with serum-free medium at the concentration based on the protein of each group, and the final 500 ⁇ l was incubated for 48 hours. After incubation, attached recipient cells were collected with a cell scraper (90020) and automated Western blotting (ProteinSimple) was performed.
  • HEK293FT cells were treated with 1 ml of a transfection reagent containing 60 ⁇ g of pcDNA3.1-eGFP + 240 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular particles were separated and obtained from the 10,000 g centrifugation supernatant, small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres were separated and obtained from the 167,000 g ultracentrifugation pellet, and supermeres were separated and obtained from the 367,000 g pellet.
  • TOPscriptTM EZ005S, Enzynomics
  • Example 7-1 An experiment was conducted to compare the endogenous functional substance delivery capabilities of the supermere, exomere, and small extracellular vesicles obtained in Example 7-1.
  • HEK293FT cells were treated with 1 ml of a transfection reagent containing 60 ⁇ g of pcDNA3.1-eGFP + 240 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres from the 167,000 g ultracentrifugation pellet, and supermeres from the 367,000 g pellet.
  • HEK293FT cells which are recipient cells, were seeded in a 24-well plate at 2.8 X 10 5 cells/well (500 ⁇ l of cell culture medium), and the supernatant was aspirated after 24 hours and washed once with DPBS. Each sample was mixed with serum-free medium to make a final volume of 500 ⁇ l and incubated for 48 hours. After that, 100 ⁇ l of 1X RIPA + PIC was added and the attached recipient cells were collected using a cell scraper (90020). The obtained cell lysate was vortexed four times every 5 minutes and centrifuged at 16,000 g for 20 minutes. The supernatant was collected and automated Western blotting (ProteinSimple) was performed. Protein expression was confirmed using eGFP (1:100, CAB4211) by loading 3 ⁇ g of cell lysate.
  • Example 8 Confirmation of intracellular internalization of Supermere via the lipid raft pathway.
  • HEK293FT cells were treated with 1 ml of a transfection reagent containing 30 ⁇ g of pcDNA3.1 + 120 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres from the 167,000 g ultracentrifugation pellet, and supermeres from the 367,000 g pellet.
  • Cy5.5-NHS ester (Lumiprobe) was mixed at a ratio of 1 mg/ml: 1 ⁇ g/ml, pipetted, and incubated overnight at 4°C to induce the fluorescence-supermere reaction. Unbound fluorescent dye was removed using a Zeba TM Spin Desalting column (40K MWCO, Thermo Fisher Scientific).
  • HEK293FT cells which are recipient cells, were seeded at 2.5 10 5 cells/well in a 24-well plate containing serum-free medium (500 ⁇ l) and incubated at 37 °C for 24 h. After treatment with three inhibitors: CPZ (Chloropromazine, clathrin-mediated endocytosis inhibitor) 10 ⁇ M, EIPA (5-(N-Ethyl-N-isopropyl)-Amiloride, macropinocytosis inhibitor) 50 ⁇ M, and M ⁇ CD (Methyl- ⁇ -Cyclodextrin, lipid raft-mediated endocytosis inhibitor) 10 mM, the cells were incubated for 30 min at 37 °C.
  • CPZ Chloropromazine, clathrin-mediated endocytosis inhibitor
  • EIPA 5-(N-Ethyl-N-isopropyl)-Amiloride, macropinocytosis inhibitor
  • M ⁇ CD Metal- ⁇ -Cyclod
  • the cells were washed with DPBS and replaced with serum-free DMEM. After incubation with 50 ⁇ g/ml of Cy5.5-labeled SuperMeer in each well, the relative MFI of Cy-5.5 was measured after 30 minutes, 3 hours, and 24 hours.
  • Fig. 13 shows the results of confirming Cy5.5 expression in recipient cells 30 minutes, 3 hours, and 24 hours after treating recipient cells with each drug and supermere.
  • Fig. 13c shows the change in the proportion of cells containing supermere stained with Cy5.5 for 24 hours after treating recipient cells with each drug and supermere.
  • HEK293FT cells were treated with 1 ml of a transfection reagent containing 30 ⁇ g of pcDNA3.1 + 120 ⁇ g of PEI mixed in serum-free medium, and after 6 hours, the medium was replaced with serum-free medium, and the supernatant was obtained 48 hours later.
  • the obtained supernatant was pretreated and concentrated according to the collection method described in Example 1, and small extracellular vesicles were separated and obtained from the 150,000 g ultracentrifugation pellet, exomeres from the 167,000 g ultracentrifugation pellet, and supermeres from the 367,000 g pellet.
  • HEK293FT cells which are recipient cells, were seeded at 2.8 ⁇ 10 5 cells/well in 6-well or 24-well plates (500 ⁇ l of medium: DMEM, high-concentration glucose + 10% FBS + 1% AA), and after 24 hours, the supernatant was aspirated and washed once with DPBS. Thereafter, groups with the conditions shown in Table 3 below were prepared, respectively.
  • Each prepared group was incubated at room temperature for 15 minutes, mixed with the above medium to make a final volume of 500 ⁇ l, and incubated for 48 hours. After that, 100 ⁇ ⁇ l of 1X RIPA + PIC was added, adherent cells were collected using a cell scraper (90020), and the obtained cell lysate was vortexed every 5 minutes, repeated 4 times, and centrifuged at 16,000 g for 20 minutes. Automated Western blotting (ProteinSimple) was performed with the obtained supernatant.
  • Example 11 Comparison of gene delivery capabilities of supermembrane and lipid nanoparticles
  • mice small extracellular vesicles and supermems were isolated and obtained from HEK293FT cells according to the collection method described in Example 1. Then, C57BL/6, male, 7-week-old mice were prepared and fed alfalfa-free feed, and samples as shown in Table 2 below were prepared, and fasting was performed one day before sample injection.
  • mice were intravascularly injected into mice, and 18 hours later, the mice were sacrificed and their organs were removed.
  • the fluorescence values of the target proteins translated in vivo were measured using an IVIS spectrum (Caliper Life Sciences, IVIS® Lumina Series III).
  • Lipid nanoparticles which are mainly used as mRNA delivery vehicles, have the problem of accumulating in large quantities in the liver and spleen and not being systematically distributed in the body. Therefore, SuperMere also conducted an in vivo distribution experiment to confirm whether this side effect exists.
  • small extracellular vesicles and supermembranes were isolated from HEK293FT cells according to the collection method described in Example 1, and 1 ⁇ g of sulfo-Cyanine5.5 NHS ester (Cy5.5; Lumiprobe, 17320) was mixed per 100 ⁇ g of small extracellular vesicles and supermembranes, respectively, and incubated overnight at 4°C. Unbound Cy5.5 was removed using Zeba TM Spin Desalting Columns (A57761), and fluorescence values were standardized between samples using a microplate reader.
  • mice C57BL/6 male, 7-week-old mice were fed an alfalfa-free diet and fasted the day before sample injection.
  • Free dye 200 ⁇ l
  • Cy5.5-labeled small extracellular vesicles 200 ⁇ g
  • Cy5.5-labeled supermere 200 ⁇ g
  • small extracellular vesicles, exomeres, and supermeres were isolated from human bone marrow stem cells according to the collection method described in Example 1, and mixed with 1 ⁇ g of sulfo-Cyanine5.5 NHS ester (Cy5.5; Lumiprobe, 17320) per 100 ⁇ g of small extracellular vesicles, exomeres, or supermeres, followed by incubation overnight at 4°C. Unbound Cy5.5 was removed using Zeba TM Spin Desalting Columns (A57761), and fluorescence values were standardized between samples using a microplate reader.
  • mice C57BL/6, male, 7-week-old mice were fed an alfalfa-free diet and fasted the day before sample injection.
  • Free dye 100 ⁇ l
  • Cy5.5-labeled small extracellular vesicles 100 ⁇ l
  • Cy5.5-labeled exomere 100 ⁇ g
  • Cy5.5-labeled supermere 100 ⁇ g
  • mice were sacrificed and organs (brain, heart, lung, liver, kidney, spleen, intestine, bladder) were removed. The removed organs were measured with IVIS spectrum (Caliper Life Sciences, IVIS® Lumina Series III) to determine the Cy5.5 fluorescence values remaining in the organs.
  • Example 13 Confirmation of selective loading of target substances into supermere through supermere marker-functional substance fusion
  • the supermere was isolated and obtained from HEK293FT cells according to the collection method described in Example 1.
  • HEK293FT cells were treated with a transfection reagent (1 ml) containing pcDNA3.1-TGFBI-Myc (60 ⁇ g), pcDNA3.1-T7-TGFBI-Myc (60 ⁇ g), and PEI (240 ⁇ g), respectively, and the medium was replaced with serum-free medium after 6 hours, and the supernatant was collected after 48 hours.
  • the plasmids used were constructed by cloning a polynucleotide encoding the TGFBI-Myc protein (SEQ ID NO: 6) into the pcDNA3.1 vector (ThermoFisher) and a polynucleotide encoding the T7-TGFBI-Myc protein (SEQ ID NO: 7) into the pcDNA3.1 vector (ThermoFisher).
  • the cell supernatant was centrifuged at 3000 g for 5 minutes and filtered through a 0.45 ⁇ m filter (Sartolab RF500 PES). After concentration with Amicon (10 kDa filter, UFC901024, Merck), the supernatant was centrifuged at 10,000 g for 30 minutes. Subsequently, the supernatant was ultracentrifuged at 150,000 g for 90 minutes at 4 °C (Ultracentrifuge, Beckman), and the supernatant was transferred to a UC tube (355655) and centrifuged at 167,000 g for 16 hours.
  • Amicon 10 kDa filter, UFC901024, Merck
  • the supernatant was then transferred to a 70Ti rotor high speed tube (355618) and centrifuged at 367,000 g for 16 hours. Supermere was then isolated from the resulting pellet using 1X PBS-PIC (stored on ice for at least 20 minutes, then briefly centrifuged for 2 minutes).
  • the T7-TGFBI-Myc supermembrane showed a protein expression pattern similar to that of the TGFBI-Myc supermembrane (Fig. 21).

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Abstract

La présente invention porte sur une composition pour l'administration de substances fonctionnelles et ses utilisations. La composition pour l'administration de substances fonctionnelles à l'aide de particules extracellulaires non vésiculaires selon la présente invention peut charger efficacement des matériaux génétiques tels que des protéines, de l'ARNm et/ou de l'ADN plasmidique et les administrer efficacement à des cellules, et peut ainsi être utilisée en tant que composition pharmaceutique ou composition cosmétique fonctionnelle pour le traitement de maladies.
PCT/KR2025/004755 2024-04-08 2025-04-08 Composition pour l'administration de substance fonctionnelle et ses utilisations Pending WO2025216537A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019109077A1 (fr) * 2017-12-01 2019-06-06 Cornell University Nanoparticules et sous-ensembles d'exosomes distincts pour la détection et le traitement du cancer
WO2022263500A1 (fr) * 2021-06-17 2022-12-22 Qiagen Gmbh Procédé d'isolement de miarn non vésiculaire
WO2023060066A2 (fr) * 2021-10-08 2023-04-13 Vanderbilt University Nanoparticules supermères et procédés d'isolement et d'utilisation de celles-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019109077A1 (fr) * 2017-12-01 2019-06-06 Cornell University Nanoparticules et sous-ensembles d'exosomes distincts pour la détection et le traitement du cancer
WO2022263500A1 (fr) * 2021-06-17 2022-12-22 Qiagen Gmbh Procédé d'isolement de miarn non vésiculaire
WO2023060066A2 (fr) * 2021-10-08 2023-04-13 Vanderbilt University Nanoparticules supermères et procédés d'isolement et d'utilisation de celles-ci

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JEPPESEN, D. K. ET AL.: "Extracellular vesicles and nanoparticles: emerging complexities", TRENDS IN CELL BIOLOGY, vol. 33, no. 8, 2023, pages 667 - 681, XP087358968, DOI: 10.1016/j.tcb.2023.01.002 *
ZHANG, Q. ET AL.: "Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets", NATURE CELL BIOLOGY, vol. 23, 2021, pages 1240 - 1254, XP037646087, DOI: 10.1038/s41556-021-00805-8 *

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