Extracellular vesicle derived from mitochondria-targeting gram-positive bacteria and recombinant gram-positive bacteria for preparing same

Abstract

The present invention relates to Gram-positive bacteria-derived extracellular vesicles (EV) having mitochondria-targeting properties and selectivity, and a use thereof. In addition, the present invention relates to a recombinant Gram-positive bacterium strain having improved extracellular vesicle-producing ability. According to the present invention, Gram-positive bacteria-derived extracellular vesicles can be efficiently produced, and active substances such as proteins and nucleic acids can be selectively delivered to mitochondria.

Description

Mitochondria-targeting extracellular vesicles derived from Gram-positive bacteria and recombinant Gram-positive bacteria for their preparation

[0001] Cross-reference of related applications

[0002] This application claims priority to Korean Patent Application No. 2024-0156636 filed on November 6, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence list

[0004] The present application includes a sequence list, which has been submitted electronically in XML format and is incorporated by reference into the present application. A copy of the sequence list created on November 5, 2025, is named KC25130SEQ.xml and has a size of 163 kilobytes.

[0005] The present invention relates to an extracellular vesicle (EV) derived from Gram-positive bacteria that selectively targets mitochondria and its uses. Additionally, the present invention relates to recombinant Gram-positive bacteria with enhanced extracellular vesicle production capacity.

[0006] Mitochondria are intracellular organelles with a structure consisting of an outer membrane, an inner membrane, and a matrix; they possess their own DNA and synthesize proteins necessary for their functions. The primary function of mitochondria is to generate usable energy through oxidative phosphorylation, and the reactive oxygen species (ROS) produced during this process regulate intracellular signaling and oxidative damage. Additionally, proteins within mitochondria regulate cell fate by controlling apoptosis signals. Furthermore, they possess functions such as regulating intracellular calcium signaling, hormone synthesis, and inflammatory responses (Signal Transduction Target Therapy, 2023, 8(1): 333).

[0007] Given the numerous functions mitochondria perform within cells, mitochondrial dysfunction leads to many diseases. Representative diseases caused by mitochondrial dysfunction include neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as well as cancer and diabetes (Nature Reviews Disease Primers, 2016, 2(1): 1-22). Two main methods are being investigated to treat mitochondrial dysfunction. First, there is a method of delivering a protein that treats the dysfunction to the mitochondria or delivering its gene into the mitochondria to express the protein through transcription and decoding processes within the mitochondria. Additionally, there is a method of delivering genetic material to the nucleus to express the target protein in the cytoplasm, or delivering the target protein to the cytoplasm and transporting it to the mitochondria using mitochondrial signaling peptides (KoreaMed Synapse, 2007, 31(3): 187-192).

[0008] However, proteins involved in mitochondrial function are highly hydrophobic, making it difficult for them to move into the interior via protein transport mechanisms, resulting in low therapeutic efficiency. Consequently, research is increasingly being conducted on methods to directly deliver substances into the mitochondria. Liposomes and synthetic polymers, widely known as existing gene therapy delivery vehicles, are being discussed as potential delivery vehicles for treating mitochondrial dysfunction (U.S. Patent Publication No. 2011-0197294, U.S. Patent Publication No. 2008-0138392). However, research results to date indicate that when using these existing gene delivery vehicles, most substances are delivered primarily to the area around the nucleus, meaning the actual amount delivered to the mitochondria is extremely small.

[0009] Therefore, to solve the above problem, a method capable of selectively delivering substances to mitochondria is required.

[0010] Prior art literature

[0011] Patent documents

[0012] (Patent Document 1) U.S. Published Patent No. 2011-0197294

[0013] (Patent Document 2) U.S. Published Patent No. 2008-0138392

[0014] Non-patent literature

[0015] (Non-patent literature 1) Signal Transduction Target Therapy, 2023, 8(1):333

[0016] (Non-patent literature 2) Nature Reviews Disease Primers, 2016, 2(1):1-22

[0017] (Non-patent literature 3) KoreaMed Synapse, 2007, 31(3):187-192

[0018] The present invention aims to solve one or more of the problems of the aforementioned prior art.

[0019] One objective of the present invention is to provide an extracellular vesicle derived from Gram-positive bacteria or a composition containing the same for the delivery of an active ingredient to mitochondria.

[0020] Another objective of the present invention is to provide recombinant Gram-positive bacteria with improved extracellular vesicle production capacity compared to unmodified Gram-positive bacteria.

[0021] Another objective of the present invention is to provide a method for obtaining an extracellular vesicle or an extracellular vesicle loaded with an active substance.

[0022] Another objective of the present invention is to provide a method for delivering an active substance to mitochondria using an extracellular vesicle or an extracellular vesicle loaded with an active substance.

[0023] Another objective of the present invention is to provide a use for extracellular vesicles or extracellular vesicles loaded with active substances for the prevention or treatment of cellular energy deficiency or mitochondrial dysfunction.

[0024] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become more apparent from the following description and will be realized by the means and combinations thereof described in the claims.

[0025] A representative configuration of the present invention for achieving the above objective is as follows.

[0026] In one aspect of the present invention, a composition comprising a Gram-positive bacterium-derived extracellular vesicle for selective delivery of an active ingredient to mitochondria is provided.

[0027] In some embodiments, the Gram-positive bacteria may belong to the genus Lacticaseibacillus or Bacillus.

[0028] In some embodiments, the Gram-positive bacteria may be a Bacillus subtilis species complex.

[0029] In some embodiments, the Gram-positive bacteria may be Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Lacticaseibacillus paracasei.

[0030] In some embodiments, the active ingredient may exist in a form that forms a complex with an extracellular vesicle or is loaded thereon.

[0031] In some embodiments, extracellular vesicles can penetrate the cell membrane and selectively move into mitochondria.

[0032] In some embodiments, extracellular vesicles derived from Gram-positive bacteria may have an average particle size in the range of 1 nm to 300 nm.

[0033] In some embodiments, the active ingredient may be a polypeptide, a nucleic acid, or a combination thereof.

[0034] In some embodiments, the active ingredient may be an ingredient for preventing, improving, or supplementing mitochondrial dysfunction or cellular energy deficiency.

[0035] In some embodiments, the active ingredient may be superoxide dismutase (SOD) or a nucleic acid encoding it.

[0036] In some embodiments, the active ingredient may be a superoxide dismutase derived from Bacillus velezensis.

[0037] In some embodiments, the composition may be a food or cosmetic composition.

[0038] In some embodiments, the composition may be a pharmaceutical composition for the prevention or treatment of cellular energy deficiency or mitochondrial dysfunction.

[0039] In another aspect of the present invention, a method for preparing a composition as described herein is provided, comprising the steps of culturing a Gram-positive bacterial strain and isolating extracellular vesicles from the culture.

[0040] In some embodiments, a method for preparing a composition as described herein is provided, comprising the steps of culturing a Gram-positive bacterial strain and isolating extracellular vesicles from the culture, wherein the Gram-positive bacterial strain is genetically engineered to produce an active ingredient required to be delivered to intracellular mitochondria, or further comprising the step of mixing the isolated Gram-positive bacterial-derived extracellular vesicles with the active ingredient required to be delivered to intracellular mitochondria.

[0041] In some embodiments, delivery to intracellular mitochondria may be performed in vitro, in vitro, or in vivo.

[0042] In some embodiments, the step of loading an active ingredient onto an extracellular vesicle derived from Gram-positive bacteria may be performed in vitro, extracellularly, or intracellularly.

[0043] In another aspect of the present invention, a recombinant Gram-positive bacterium is provided that has an improved ability to produce extracellular vesicles compared to an unmodified Gram-positive bacterium.

[0044] In some embodiments, the recombinant Gram-positive bacteria may be a strain belonging to the genus Lacticase Bacillus or Bacillus.

[0045] In some embodiments, the recombinant Gram-positive bacteria may be strains belonging to the Bacillus subtilis species complex.

[0046] In some embodiments, the recombinant Gram-positive bacteria may be Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus paracasei lacticasei strains.

[0047] In some embodiments, the recombinant Gram-positive bacteria may have one or more genes selected from the group consisting of AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAC, EpsE, Xpf, lytC, PBSX, SP-β, cotE, skin, sigE, hlyⅢ, wprA, Saf-coxA, and spoIVA deleted compared to the wild type.

[0048] In some embodiments, the recombinant Gram-positive bacteria may have one or more genes selected from the group consisting of AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, EpsE, and wprA deleted compared to the wild-type strain.

[0049] In some embodiments, the recombinant Gram-positive bacteria may be a recombinant Gram-positive bacterial strain that additionally includes a foreign gene encoding an active substance for delivery to mitochondria.

[0050] In another aspect of the present invention, a method for delivering an active substance to mitochondria is provided, comprising the step of administering a composition as disclosed herein to a target.

[0051] In another aspect of the present invention, a method for preventing, improving, or treating cellular energy deficiency or mitochondrial dysfunction is provided, comprising the step of administering a composition as disclosed herein to a target.

[0052] In some embodiments, mitochondrial dysfunction or cellular energy deficiency may be selected from the group consisting of Leigh Syndrome, MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes), Leber Hereditary Optic Neuropathy (LHON), Kearns-Sayre Syndrome (KSS), MERRF (Myoclonic Epilepsy with Ragged-Red Fibers), and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP).

[0053] In another embodiment of the present invention, a use is provided for preventing, improving, or treating cellular energy deficiency or mitochondrial dysfunction of a composition as disclosed in this specification.

[0054] The Gram-positive bacterium-derived extracellular vesicle of the present invention exhibits excellent cell permeability and mitochondrial targeting ability capable of selectively reaching mitochondria. By loading active ingredients, such as proteins or nucleic acids, and effectively delivering them to mitochondria, it has the effect of enhancing mitochondrial function or treating damage. Due to these effects, the Gram-positive bacterium-derived extracellular vesicle of the present invention can be utilized for the delivery of active ingredients that need to be delivered to mitochondria, or for subjects with diseases involving mitochondrial dysfunction. In one embodiment of the present invention, when a fluorescent protein was loaded into an EV using recombinant Gram-positive bacteria (e.g., B. subtilis) and then treated with eukaryotic cells, the blue fluorescent protein loaded into the EV was primarily observed inside the mitochondria of the eukaryotic cells. In one embodiment of the present invention, it was confirmed that the mitochondrial membrane potential is restored when an EV generated from a recombinant Gram-positive bacterium (e.g., B. subtilis) loaded with superoxide dismutase is treated to eukaryotic cells treated with H2O2. In one embodiment of the present invention, it was confirmed that the EV of the recombinant B. subtilis can selectively deliver the loaded substance to mitochondria by observing that the blue fluorescent protein is expressed within the mitochondria of the eukaryotic cells when an EV generated from a recombinant Gram-positive bacterium (e.g., B. subtilis) loaded with DNA encoding a blue fluorescent protein is treated to eukaryotic cells.

[0055] In addition, the present invention provides a recombinant Gram-positive bacterium (e.g., B. subtilis) with improved extracellular vesicle productivity. The recombinant Gram-positive bacterium of the present invention exhibits significantly improved extracellular vesicle production capacity compared to unmodified Gram-positive bacteria. In one embodiment, it was confirmed that the recombinant Gram-positive bacterium of the present invention (e.g., B. subtilis) has an extracellular vesicle productivity of approximately 7.4 times higher than that of the wild type.

[0056] Figure 1 shows a schematic diagram for the double cross-recombination performed in Example 1.1.

[0057] Figure 2 shows the results of confirming the deletion site of B. subtilisGF#19 using agarose electrophoresis after PCR with the primers in Table 4.

[0058] Figure 3 shows an expression vector for the overexpression of the sodA2 gene. sodA2 represents the sodA2 gene; rrnBT1T2 represents a transcription termination factor; rep(pBR322) represents a replicon from pBR322 that operates in E. coli; rep(pUB110) represents a replicon from pUB110 that operates in B. subtilis; and Kan R represents the kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); Amp R represents an ampicillin resistance gene; BJ27 represents a potent promoter for B. subtilis.

[0059] Figure 4 shows an expression vector for the overexpression of the sodA2-EBFP gene. sodA2 represents the sodA2 gene; EBFP represents the EBFP gene; rrnB T1T2 represents a transcription termination factor; Rep(pBR322) represents a replicon from pBR322 that operates in E. coli; rep(pUB110) represents a replicon from pUB110 that operates in B. subtilis; and Kan R represents the kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); Amp R represents an ampicillin resistance gene; BJ27 represents a potent promoter for B. subtilis.

[0060] Figure 5 shows a schematic diagram of Mt_p2-EBFP, a DNA fragment in which an expression promoter in mitochondria and an EBFP fluorescent protein gene are combined.

[0061] Figure 6a shows the size distribution and concentration of purified EVs in a culture medium in which Bs_GF#19 was cultured.

[0062] Figure 6b shows the size distribution and concentration of purified EVs in a culture medium in which Bs_GF#19-pBE1-SODA2 was cultured.

[0063] Figure 6c shows the size distribution and concentration of purified EVs in a culture medium in which Bs_GF#19-pBE1-SODA2-EBFP was cultured.

[0064] Figure 7a shows the size distribution and concentration of GF#19 EVs into which mt-EBFP was inserted by electroporation.

[0065] Figure 7b shows the size distribution and concentration of GF#19 EVs with mt_p2-EBFP inserted by electroporation.

[0066] Figure 8a shows a fluorescence image of Vero cells treated with EV observed with HT-2H.

[0067] Figure 8b shows a fluorescence image of GM13411 cells treated with EV observed with HT-2H.

[0068] Figure 9 is a graph showing the change in mitochondrial superoxide concentration using MitoSOX after EV treatment with H2O2, EV, and SOD loaded onto Vero cells.

[0069] Figure 10a shows fluorescence images of mitochondrial membrane potential observed with HT-2H using JC1 after HeLa cells were treated with H2O2 alone and H2O2+ EV combined.

[0070] Figure 10b is a graph showing the proportion of JC1 in the fluorescence observed in Figure 9a.

[0071] Figure 11 shows a fluorescence image of GM13411 cells treated with EV observed with HT-2H.

[0072] Figure 12a shows the size distribution and concentration of purified EV in a culture medium in which L. paracasei was cultured.

[0073] Figure 12b shows the size distribution and concentration of purified EVs in a culture medium in which B. velezensis FZB42 was cultured.

[0074] Figure 12c shows the size distribution and concentration of purified EVs in a culture medium in which B. velezenesis GF423 was cultured.

[0075] Figure 13 shows a fluorescence image of Vero cells treated with EV observed with HT-2H.

[0076] The following detailed description of the present invention will be described with reference to specific drawings (where drawings are available) regarding specific embodiments in which the present invention may be practiced, but the present invention is not limited thereto and is limited only by the appended claims to all scopes identical or equivalent to those described in the claims. It should be understood that various embodiments of the present invention are different but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be modified from one embodiment to another or realized by combining multiple embodiments without departing from the technical spirit and scope of the present invention. Technical and academic terms used herein have the same meaning as commonly used in the field to which the present invention belongs, unless otherwise defined. For the purposes of interpreting this specification, the following definitions shall apply, and terms expressed in the singular form shall be interpreted as also referring to the plural form (i.e., at least one) unless inappropriate in the context, and vice versa.

[0077] Gram-positive bacteria-derived extracellular vesicles that selectively target mitochondria

[0078] The present invention is based on the discovery that extracellular vesicles derived at least partially from Gram-positive bacteria (e.g., Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus lacticasei paracasei) can carry active ingredients, such as polypeptides or nucleic acids, penetrate the cell membrane, and selectively deliver the active ingredients to intracellular mitochondria. Accordingly, the present invention provides extracellular vesicles derived from Gram-positive bacteria (e.g., Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus lacticasei paracasei) that can selectively move to intracellular mitochondria in vivo or in vitro while carrying active ingredients, compositions comprising the same, and uses and methods thereof for delivering active ingredients to intracellular mitochondria. Extracellular vesicles derived from Gram-positive bacteria of the present invention (e.g., Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus lacticasei paracasei) can be consistently produced having desired characteristics (e.g., cell membrane permeability, mitochondrial targeting, and a lipid bilayer structure capable of forming complexes with active substances or loading active substances). In particular, the extracellular vesicles of the present invention, which can selectively move into intracellular mitochondria, can be utilized as mitochondrial-targeted drug delivery systems capable of delivering active ingredients for the prevention or treatment of diseases or disorders associated with mitochondrial dysfunction or cellular energy deficiency.

[0079] In one aspect of the present invention, a composition comprising extracellular vesicles derived from Gram-positive bacteria for the selective delivery of an active ingredient to mitochondria is provided. As used herein, the term "extracellular vesicle" refers to a nano-sized particle having a lipid bilayer structure obtained from a living cell, and may be used interchangeably with terms referring to particles of similar structure, such as exosomes, microvesicles, polyvesicles, or extracellular vesicle-like particles.

[0080] In some embodiments, the extracellular vesicles of the present invention may be derived from Gram-positive bacteria.

[0081] In some embodiments, the extracellular vesicles of the present invention are microorganisms belonging, for example, of the genus Bacillus, preferably microorganisms belonging to the Bacillus subtilis species complex (Int J Syst Evol Microbiol (2009) 59: 2429-2436), such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus pumilus, Bacillus sonorensis, Bacillus tequilensis, Bacillus vallismortis, or Bacillus velezensis, more preferably Bacillus subtilis Or it may be derived from Bacillus velezensis. In some embodiments, the extracellular vesicles of the present invention may be derived from microorganisms belonging to the genus Lacticaseibacillus, such as Lacticaseibacillus rhamnosus, Lacticaseibacillus zeae, Lacticaseibacillus casei, Lacticaseibacillus chiangmaiensis, or Lacticaseibacillus paracasei, more preferably from Lacticaseibacillus paracasei.In some embodiments, the extracellular vesicles of the present invention may be derived from the Bacillus subtilis strain deposited under accession number KCTC 3135, the Bacillus velezensis strain deposited under accession number KCTC 13222 BP, the Bacillus velezensis strain deposited under accession number KCTC 13227 BP, the Bacillus velezensis strain deposited under accession number KCTC 15552 BP, the Bacillus velezensis strain deposited under accession number KCTC 1660, or the Lacticasei-Bacillus paracasei strain deposited under accession number KCTC 3165. In some embodiments, the extracellular vesicles of the present invention may be isolated extracellular vesicles. In some embodiments, the extracellular vesicles of the present invention may be extracellular vesicles isolated from a culture of Bacillus, such as Bacillus subtilis or Bacillus velezensis. In some embodiments, the extracellular vesicle of the present invention may be an extracellular vesicle isolated from a culture of Lacticase Bacillus, such as Lacticase Bacillus paracasei.

[0082] In some embodiments, the extracellular vesicle of the present invention has a lipid bilayer structure capable of penetrating a cell membrane in vivo or in vitro and moving into mitochondria, and specifically a lipid bilayer structure capable of forming a complex with an active substance or containing an active substance. Without being bound by theory, the extracellular vesicle of the present invention, which forms a complex with an active substance or contains an active substance, maintains a substantially intact lipid bilayer structure in vivo or in vitro so as to penetrate a cell membrane and move into mitochondria, allowing the active substance to function within the mitochondria.

[0083] In some embodiments, the average particle size of the extracellular vesicles of the present invention is nanoscale, for example, in the range of about 1 nm to about 300 nm, about 10 nm to about 250 nm, about 20 nm to about 200 nm, about 30 nm to about 160 nm, about 40 nm to about 140 nm, about 50 nm to about 120 nm, about 70 nm to about 100 nm, about 80 nm to about 90 nm, about 82 nm to about 88 nm, about 83 nm to about 87 nm, or about 84 nm to about 87 nm. In a preferred embodiment, the average particle size of the extracellular vesicles of the present invention is in the range of about 80 nm to about 90 nm, more preferably about 84 nm or about 87 nm. In another embodiment, the average particle size of the extracellular vesicle of the present invention is within the range of about 80.0 nm, about 81.0 nm, about 82.0 nm, about 83.0 nm, about 83.5 nm, about 84.0 nm, about 84.5 nm, about 85.0 nm, about 85.0 nm, about 86.0 nm, about 86.5 nm, about 87.0 nm, about 87.5 nm, about 88.0 nm, about 89.0 nm, about 90.0 nm, about 50 to 100 nm, 60 to 90 nm, or 70 to 85 nm.

[0084] In this specification, the term “active ingredient” may be a substance introduced into a cell to enhance, inhibit, or alter cell function, specifically a substance that enhances, inhibits, or alters the function of mitochondria within the cell, or a substance capable of improving cellular energy deficiency. In some embodiments, the active ingredient may be, but is not limited to, proteins, polypeptides, nucleic acids (e.g., DNA or RNA fragments), carbohydrates, lipids, organic materials, inorganic materials, small molecule compounds (e.g., metal compounds or non-metal compounds). In some embodiments, the active ingredient may include polypeptides, nucleic acids, or combinations thereof.

[0085] In some embodiments, the active ingredient may be a polypeptide-based therapeutic, such as one that enhances, inhibits, or alters the function of intracellular mitochondria or improves cellular energy deficiency. As an example, the active ingredient may be superoxide dismutase. Specifically, the active ingredient may be a Bacillus-derived superoxide dismutase. More specifically, the active ingredient may be a superoxide dismutase derived from microorganisms belonging to the Bacillus subtilis species complex (Int J Syst Evol Microbiol (2009) 59: 2429-2436), such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus pumilus, Bacillus sonorensis, Bacillus tequilensis, Bacillus vallismortis, or Bacillus velezensis. More specifically, the active ingredient may be a superoxide dismutase derived from Bacillus velezensis. In some embodiments, Bacillus velezensis may be the Bacillus velezensis strain deposited under accession number KCTC 13222 BP, the Bacillus velezensis strain deposited under accession number KCTC 13227 BP, or the Bacillus velezensis strain deposited under accession number KCTC 15552 BP.More specifically, the active ingredient may be a superoxide dismutase polypeptide comprising the amino acid sequence of SEQ ID NO. 4 or an amino acid sequence having at least 70% identity with said sequence (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%).

[0086] In some embodiments, the active ingredient may be a DNA-based therapeutic or an RNA-based therapeutic, such as a DNA-based therapeutic or an RNA-based therapeutic that enhances, inhibits, or alters the function of intracellular mitochondria or improves cellular energy deficiency. Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or nonviral synthetic DNA vectors, closed linear divalent DNA (ceDNA / CELiD), plasmids, Bacmids, DNA vectors, minimally immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (DNA vectors closed by linear covalent bonds), dumbbell DNA minimal vectors ("dumbbell DNA"), gDNA, cDNA, pDNA, or combinations thereof. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA, oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer-substrate dsRNA, small hairpin RNA (shRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, viral RNA vectors, guide RNA, or combinations thereof. Additionally, the active substance may be PNA, BNA, aptamers, cyclic dinucleotides, or combinations thereof.

[0087] In some embodiments, the DNA-based therapeutic may include a mitochondria-specific promoter for protein expression in mitochondria. Non-limiting examples of mitochondria-specific promoters include LSP (light-strand promoter), HSP1 (heavy-strand promoter 1), or HSP2.

[0088] In some embodiments, the active substance may form a complex with the extracellular vesicle of the present invention or be contained within the extracellular vesicle.

[0089] In some embodiments, the active substance may be generated from a foreign gene introduced into a Gram-positive bacterial cell from which the extracellular vesicle of the present invention is derived. In some embodiments, the active substance may be generated independently of the Gram-positive bacterial cell from which the extracellular vesicle of the present invention is derived. For example, an active substance such as a polypeptide may be generated from a foreign gene introduced into a Gram-positive bacterial cell from which the extracellular vesicle of the present invention is derived and loaded onto the extracellular vesicle of the present invention (forming a complex with the extracellular vesicle or being contained within the extracellular vesicle and secreted outside the Gram-positive bacterial cell). For example, an active substance such as a nucleic acid may be generated or synthesized independently of the Gram-positive bacterial cell and loaded onto the extracellular vesicle derived from the Gram-positive bacterial cell of the present invention in vitro or in vitro (forming a complex with the extracellular vesicle derived from the Gram-positive bacterial cell of the present invention or being contained within the extracellular vesicle derived from the Gram-positive bacterial cell of the present invention).

[0090] In another aspect of the present invention, a method for producing extracellular vesicles derived from Gram-positive bacteria as disclosed herein is provided. Specifically, a method for producing extracellular vesicles derived from Gram-positive bacteria as disclosed herein is provided, comprising the steps of culturing a Gram-positive bacterial strain (e.g., Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Lacticasei-Bacillus paracasei strain) and isolating extracellular vesicles from the obtained culture.

[0091] In some embodiments, the culture may be the supernatant from which cells have been removed.

[0092] In some embodiments, the separation of extracellular vesicles may include the step of filtering the culture by a tangential flow filtration (TFF) method, and optionally filtering it through a hollow fiber membrane.

[0093] In some embodiments, the method for preparing extracellular vesicles derived from Gram-positive bacteria described herein may further include the step of further sterilizing the filtrate (e.g., filtration sterilization).

[0094] In some embodiments, the Gram-positive bacterial strain may be a recombinant Gram-positive bacterial strain containing a gene encoding an active substance to be loaded onto an extracellular vesicle derived from Gram-positive bacteria described herein. In this case, an extracellular vesicle loaded with the active substance is obtained.

[0095] In some embodiments, the Gram-positive bacterial strain may be a recombinant Gram-positive bacterial strain engineered for the efficient production of the Gram-positive derived extracellular vesicles described herein. A detailed description of the engineered recombinant Gram-positive bacterial strain is provided below.

[0096] In some embodiments, the manufacturing method further includes the step of mixing the obtained extracellular vesicles with the active substance. This step is performed to load the active substance onto the extracellular vesicles in vitro.

[0097] In another aspect of the present invention, a system for selective delivery of an active substance to mitochondria is provided, comprising a composition described herein. In another aspect, a method for delivering an active substance to mitochondria is provided, comprising the step of administering the composition described herein to a target. In some embodiments, the target may be a mammal with impaired mitochondrial function requiring administration of the active substance, e.g., primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0098] In another aspect of the present invention, a pharmaceutical composition comprising the composition described herein and a pharmaceutically acceptable excipient or carrier is provided. In another aspect, a cosmetic composition comprising the composition described herein is provided. In another aspect, a food product comprising the composition described herein is provided.

[0099] In some embodiments, the pharmaceutical, food, or cosmetic composition may be intended to prevent, improve, or treat cellular energy deficiency or to prevent, improve, or treat mitochondrial dysfunction. Mitochondrial dysfunction refers to any disease caused by mitochondrial function not operating normally or deviating from normal levels.

[0100] In some embodiments, mitochondrial dysfunction or cellular energy deficiency may be selected from the group consisting of Leigh Syndrome, MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes), Leber Hereditary Optic Neuropathy (LHON), Kearns-Sayre Syndrome (KSS), MERRF (Myoclonic Epilepsy with Ragged-Red Fibers), and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP), but is not limited thereto.

[0101] The term "treatment" means any measure that provides benefit to an individual to whom the extracellular vesicle of the present invention may be administered, including the treatment, alleviation, or improvement of any disease state or symptom regulated by the active ingredient contained in the extracellular vesicle of the present invention.

[0102] The term "prevention" refers to any measure that provides benefit to an individual to whom the extracellular vesicle of the present invention may be administered, including the prevention of any disease state or symptom regulated by the active ingredient contained in the extracellular vesicle of the present invention or the prevention of exacerbation thereof. Depending on the characteristics of the disease, the meanings of "treatment" and "prevention" may partially overlap.

[0103] The pharmaceutical composition according to the present invention may further comprise conventional and non-toxic pharmaceutically acceptable additives that are formulated into a formulation according to conventional methods. For example, the pharmaceutical composition may further comprise pharmaceutically acceptable carriers, diluents, excipients, stabilizers, preservatives, buffers, sweeteners, etc.

[0104] Suitable carriers, excipients, or diluents include, for example, carbohydrates, waxes, water-soluble and / or swelling polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc. The specific carrier, diluent, or excipient used may be selected according to the means and purpose for which the extracellular vesicles of the present invention are applied. The solvent is generally selected based on solvents recognized by a person skilled in the art as safe for administration to mammals (GRAS).

[0105] Examples of additives used in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, minerals such as silicon dioxide, etc.

[0106] The composition of the present invention may be administered orally (e.g., tablets, pills, powders, capsules, syrups, or emulsions) or parenterally. When administered parenterally, it may be administered into the ventricles, dura mater, abdominal cavity, rectum, subcutaneously, nasally, percutaneously, intravenously, intra-arterially, intramuscularly, or thoracically. Additionally, it may be administered locally at the site where the disease has occurred.

[0107] The composition of the present invention may preferably be formulated as an oral administration formulation. In this case, solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid formulations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the complex composition. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and various excipients, for example, humectants, sweeteners, flavorings, preservatives, etc., may be used in addition to commonly used simple diluents such as water and liquid paraffin.

[0108] In addition, preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. Injectable preparations may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0109] The extracellular vesicle or composition of the present invention may be administered to an individual in an effective amount, for example, an amount effective for therapeutic or prophylactic purposes or an amount effective for pharmaceutical purposes.

[0110] "A therapeutically or prophylactically effective amount" or "a pharmaceutically effective amount" refers to an amount of extracellular vesicles or a composition effective for preventing or treating a target disease, meaning an amount sufficient to prevent or treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The level of the said effective amount may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0111] The extracellular vesicles or compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, administered sequentially or simultaneously with conventional therapeutic agents, and administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without or with minimal side effects, taking into account all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

[0112] Specifically, the effective amount of extracellular vesicles in the composition of the present invention may vary depending on the age, gender, and body weight of the individual (patient), and generally, about 0.1 mg to 1,000 mg or 5 mg to about 200 mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since it may be increased or decreased depending on the route of administration, severity of the disease, gender, body weight, age, etc., the scope of the present invention is not limited thereto.

[0113] The pharmaceutical composition of the present invention may additionally include one or more active ingredients. The additional active ingredients may be known anticancer agents, treatments for neurodegenerative diseases, or treatments for mitochondrial dysfunction.

[0114] The pharmaceutical composition of the present invention can be used by formulating it into the form of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, aerosols, injectable solutions, etc., according to conventional methods.

[0115] In addition, the pharmaceutical composition of the present invention may be formulated into a preferred form depending on the method of use, and may be formulated by adopting methods known in the art to provide rapid, sustained, or delayed release of the active ingredient, particularly after administration to mammals. Specific examples of such formulations include granules, powders, syrups, liquids, aerosols, extracts, elixirs, fluid extracts, emulsions, suspensions, liquids, infusions, tablets, capsules, pills, soft or hard gelatin capsules, injectable solutions, etc.

[0116] Furthermore, the pharmaceutical composition of the present invention may preferably be formulated using a suitable method known in the art or using a method disclosed in Remington's literature (Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, PA).

[0117] In some embodiments, the food may be a health functional food or a medical food. As used herein, the terms "health functional food" or "medical food" refer to a food manufactured using raw materials or ingredients that perform beneficial functions for the human body, as defined by the Ministry of Food and Drug Safety, which maintains normal human functions or activates physiological functions to maintain or improve health; however, the term is not necessarily limited thereto and does not exclude any conventional health food in this sense. The food of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. Conventional additives include, for example, chemically synthesized additives such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon dye, licorice extract, crystalline cellulose, sorghum dye, and guar gum; and mixed preparations such as L-sodium glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar dye preparations.

[0118] The cosmetic composition of the present invention may be prepared in any formulation conventionally manufactured in the art, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray, but is not limited thereto. More specifically, it may be prepared in the form of a softening lotion, a nourishing lotion, a cream, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

[0119] Recombinant Bacillus with improved extracellular vesicle production capacity

[0120] In one aspect of the present invention, a recombinant Gram-positive bacterial strain (e.g., a recombinant Bacillus subtilis strain) is provided, which has improved extracellular vesicle production capacity compared to an unmodified Gram-positive bacterium (e.g., Bacillus subtilis). Specifically, the recombinant Gram-positive bacterial strain of the present invention may be a strain in which some genes are deleted to improve the extracellular vesicle production capacity of Bacillus subtilis.

[0121] In some embodiments, the recombinant Gram-positive bacterial strain may be a strain in which the yield or stability of extracellular vesicles is improved and / or the purification process of extracellular vesicles is improved compared to an unmodified Gram-positive bacterial strain.

[0122] In some embodiments, the recombinant Gram-positive bacterial strain may be a microorganism belonging to, for example, the genus Bacillus, preferably a microorganism belonging to the Bacillus subtilis species complex (Int J Syst Evol Microbiol (2009) 59: 2429-2436), such as Bacillus amyloliquefaciens, Bacillus atropheres, Bacillus licheniformis, Bacillus mozavensis, Bacillus pumilus, Bacillus sonorensis, Bacillus tequilensis, Bacillus vallismortis, or Bacillus velezensis, more preferably a Bacillus subtilis or Bacillus velezensis strain. In some embodiments, the recombinant Gram-positive bacterial strain may be a microorganism belonging to the genus Lacticaseibacillus, such as Lacticaseibacillus rhamnosus, Lacticaseibacillus zeai, Lacticaseibacillus casei, Lacticaseibacillus chiangmaiensis, or Lacticaseibacillus paracasei, more preferably a Lacticaseibacillus paracasei strain.

[0123] In some embodiments, the gene deleted to improve the production capacity of extracellular vesicles may be one or more selected from the group consisting of AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAC, EpsE, Xpf, lytC, PBSX, SP-β, cotE, skin, sigE, hlyⅢ, wprA, Saf-coxA, and spoIVA. In some embodiments, the gene deleted to improve the production capacity of extracellular vesicles may be one or more selected from the group consisting of AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, EpsE, and wprA. Without being bound by theory, these genes were designed to be deleted for the removal of extracellular proteases, extracellular polysaccharides, and surfactins. In a preferred embodiment, the genes deleted to improve the production capacity of extracellular vesicles may be AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, EpsE, and wprA. In another preferred embodiment, the genes deleted to improve the production capacity of extracellular vesicles may be AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAC, EpsE, Xpf, lytC, PBSX, SP-β, cotE, skin, sigE, hlyⅢ, wprA, Saf-coxA, and spoIVA. The deletion of genes may be performed by conventional methods known in the art, such as double cross-recombination.

[0124] In some embodiments, the recombinant Gram-positive bacterial strain may additionally include a foreign gene encoding an active substance for delivery to mitochondria.

[0125] In some embodiments, recombinant Gram-positive bacterial strains may be used to produce Gram-positive derived extracellular vesicles as described herein.

[0126] All patents and references cited herein are incorporated herein by reference in their entirety. Any reference to any prior art in this specification does not constitute an acknowledgment or implied that such prior art forms part of ordinary knowledge or that it would be reasonably expected of a person skilled in the art to combine it with other prior art.

[0127] To better understand the invention described herein, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the invention in any way.

[0128] Example 1. Preparation of a strain with improved EV productivity

[0129] Example 1.1. Preparation of a strain (B. subtilisGF#19) with improved Bacillus-derived extracellular vesicle (EV) productivity

[0130] B. subtilisGF#19, a strain with enhanced EV production capacity, was prepared using Bacillus subtilisKCTC 3135 as the parent strain. B. subtilisKCTC 3135 was obtained from the Korea Culture Collection Center (KCTC) at the Korea Research Institute of Biotechnology and Bioengineering. To improve the yield, stability, and purification process of EV, the genes listed in Table 1 were removed from the genome of B. subtilisKCTC 3135.

[0131] Gene Name Protein Name Function AprE Serine Alkaline Protease (Subtilisin E) Extracellular Protease NprE Extracellular Neutral Metalloprotease Extracellular Protease Bpr Bacillus Peptidase F Extracellular Protease Epr Extracellular Serine Protease Extracellular Protease Npr B Extracellular Neutral Protease B Extracellular Protease Vpr Extracellular Serine Protease Extracellular Protease Mpr Extracellular Metalloprotease Extracellular Protease IspA Intracellular Serine Protease Intracellular Protease SrfAC Surfactin Syntase Surfactin Synthesis spoIIAC RNA Polymerase Spore-forming-Specific Sigma Factor (Sigma-F) Spore-forming EpsE Glycosyltransferase Extracellular Polysaccharide Synthesis XpfRNA Polymerase Sigma Factor PBSX Prophage Gene Transcription lytCN-acetylmuramoyl-L-alanine amidase cell wall degradation PBSX PBSX prophage prophage SP-β SP-β prophage prophage cotE spore coat protein spore formation skinskin prophage prophage sigE sigma factor sigE spore formation hlyⅢ hemolysin III - like protein unknown wprA quality control protease quality control Saf-cox As pore coat formations pore coat formationspoIV AATTPase spore formation

[0132] Gene deletion was performed using double cross-over recombination on the genome (Fig. 1). Specifically, DNA fragments (upper and lower fragments) homologous to the DNA sequences at both ends of the deletion site on the genome of B. subtilisKCTC 3135 were cloned using PCR into the vector pUCori-ts-cm, which has a temperature-sensitive replication origin. The PCR conditions used for cloning are shown in Table 2, and the nucleotide sequences of the primers used for strain preparation are presented in Table 3.

[0133] PCR Step Temperature Time Initial Denaturation 95 ℃ 30 seconds 30 cycles 95 ℃ 50 ℃ 72 ℃ 30 seconds 30 seconds 1 min / kb Final Extension 72 ℃ 10 min The DNA polymerase used for PCR is Taq DNA polymerase (NEB, USA).

[0134] 프라이머 이름프라이머 서열(5' → 3')서열번호aprEUp frag.FCTACGGGGTCTGACGCAGATATCGTAACCGAAGAACG6RTGTTGCAGACATGTTGCTGAACGCCATC7Down frag.FCAACATGTCTGCAACATATCTTGGAAAC8RGATAAGCTGTCAAACCAGAGATTGGATAGGCTATCAAG9nprEUp frag.FCTACGGGGTCTGACGCAGGGCATTACTGCACCATAATC10RGAGGTACGCCTTCAGCAGCCTGAACACC11Down frag.FGCTGAAGGCGTACCTCACGCCTTCTTCC12RGATAAGCTGTCAAACCAGTTGTCAGATTTGCATCCTTC13bprUp frag.FCTACGGGGTCTGACGCAGAGCAATTTCCAGCCCGCTTC14RGTATGATGAGGCATGAACAGCAATCCCG15Down frag.FTCATGCCTCATCATACTCAACAAGGGTG16RGATAAGCTGTCAAACCAGTTCCCAGCCGGATGTTCAAC17eprUp frag.FCTACGGGGTCTGACGCAGGCTTTCTGCCAGCCGATAGG18RTGTGTCAAAGCCGTTATGCTTGGCTCCG19Down frag.FTAACGGCTTTGACACAGCACAAACTGCC20RGATAAGCTGTCAAACCAGGTCTCCCATACATGAACGAC21nprBUp frag.FCTACGGGGTCTGACGCAGAGTAATCTTGTAGGAGGCTG22RTGTGACTGTCATATTCACCGTCGTGTGG23Down frag.FTGAATATGACAGTCACACAGTATTCCGCC24RGATAAGCTGTCAAACCAGATTAGGATACGGTCTGGCTG25vprUp frag.FCTACGGGGTCTGACGCAGATGTACGCTGAGCCGAATAG26RGGAATCACATTTGCGGAGATACGGCGTC27Down frag.FCCGCAAATGTGATTCCGGCACATCAAAC28RGATAAGCTGTCAAACCAGACCGTTTTCCGAATCTGACC29mprUp frag.FCTACGGGGTCTGACGCAGCGTTATCCCGAATGCCCGTG30RGGCTTTGTTCCTTTAGTGTCAACCACCC31Down frag.FCTAAAGGAACAAAGCCGATTCGCTCCGC32RGATAAGCTGTCAAACCAGAAATTGACTGCTCCACGCTG33srfACUp frag.FCTACGGGGTCTGACGCAGGATATGTATTACCTATCGCC34RGCCCCTTCATCCAGTTCACTGCTTCCTTG35Down frag.FGGAAGCAGTGAACTGGATGAAGGGGCTTCGCTG36RGATAAGCTGTCAAACCAGAACGCTTCGACATCACTTTC37spoIIAUp frag.FCTACGGGGTCTGACGCAGAGCGGACGATGGGAGACTGG38RGCCACCTCATGCAGCCGATCTGGAAGAG39Down frag.FGGCTGCATGAGGTGGCTGAGCGGCTCGG40RGATAAGCTGTCAAACCAGCTCATTGTTTTGGTGAGCTG41ispAUp frag.FCTACGGGGTCTGACGCAGTGCTGCTTGGCATTCATTTC42RGCCATTGAAGCAATGCCTCCGTTTGAATC43Down frag.FACGGAGGCATTGCTTCAATGGCTGCCCCTCATG44RGATAAGCTGTCAAACCAGTCAATGCTCTCGTCATATTC45epsEUp frag.FCTACGGGGTCTGACGCAGCAAGCAAATGGGCTGGGAGG46RGACAAGCCATGCTTTCTGCCAAAGTGCG47Down frag.FGAAAGCATGGCTTGTCAAGCATGAATAG48RGATAAGCTGTCAAACCAGTGTTGTATACATTCAGCAGC49xpfUp frag.FGATCCTCTACGGTTCATCCATGTCCGAAC50RTCTATGATCCTCCTCATTTTTG51Down frag.FGAGGAGGATCATAGAAAGCTTGTCATACGTTTGCC52RGAGTTTTCGTTCGGATCCTCCGCTTTTTGTTTG53lytCUp frag.FGATCCTCTACGGCCGCTTTTCAAGTTTA 54RGATTTGTCTCTTTCGATTGATTTCCTCCTTAAATG 55Down frag.FGAGGAAATCAATCGAAAGAGACAAATCTAATCACA 56RGTTTTCGTTCGGATCTTTAAGCTCAGATTGATGGT57PBSXUp frag.FGATCCTCTACGGTTCATCCATGTCCGAAC58RTCTATGATCCTCCTCATTTTTG59Down frag.FGAGGAGGATCATAGACAAAGACCATAAAAATCCCG 60RGTTTTCGTTCGGATCGGCTTCTCTTTGCCA 61SP-βUp frag.FGATCCTCTACGGGTAAAATATATACAGGT62RCTTACTACTTTTCGCTTCTTTTTTTCGT 63Down frag.FGAAAAAAAGAAGCGAAAAGTAGTAAGTATCT64RTTTCGTTCGGATCAGAAGCCGAACTGCT 65cotEUp frag.FCTCTACGGGATCCGATATTCTTGCTGGC66RCTAGTCCCTTTTTCCGGCATGCCTCCTT67Down frag.FCATGCCGGAAAAAGGGACTAGGGGAGA 68RGTTTTCGTTCGGATCCGCTTTGAGTACAAGT 69skinUp frag.FGATCCTCTACGGCAATGGTACAAATA 70RGCTTCTTCTGCAAGAGGGGTGTAT 71Down frag.FCCCTCTTGCAGAAGAAGCCGGAT 72RCGTTCGGATCTGTTTGGAGAT 73sigEUp frag.FGATCCTCTACGGGGAACAACAGATGCCT 74RAAAGGCTTTTCAATCTGACTCCTTTC75Down frag.FGGAGTCAGATTGAAAAGCCTTTAAAAC 76RGAGTTTTCGTTCGGATCGCGGAAAGTATTTGAT77hlyⅢUp frag.FGATCCTCTACGGGATCCTGGAATCTTCGAGA 78RGCTTTTCTGTTACTGGTACATTCTCCTTTCGA 79Down frag.FGAAAGGAGAATGTACCAGTAACAGAAAAGCGCA 80RGTTTTCGTTCGGATCCGACTGTATATGACGAAA 81wprAUp frag.FGATCCTCTACGGGATCC AGCTGCGCCAATCCGGT82RCGAGCACCGCTTTTTGGGTTATCCCTCCTGCAAA83Down frag.FTTTGCAGGAGGGATAAC CCAAAAAGCGGTGCTCG84RGTTTTCGTTCGGATCCCAGGCCCTCGTTTGGA85safcoxAUp frag.FGATCCTCTACGGGATCC CGATATGACCTCGTGCC86RCATAGGAGGGGAAAACGGAAGGAAGGGCTGCCGG87Down frag.FCCGGCAGCCCTTCCTTC CGTTTTCCCCTCCTATG88RGTTTTCGTTCGGATCCGGCATGGAAGAAGCTTCAG89spoIVAUp frag.FGATCCTCTACGGGATCC CATGTTCTATGTCCTCCAC90RGTCCGGGAGGGGATCACTACCGGTAGACCTCTTT91Down frag.FAAAGAGGTCTACCGGTA GTGATCCCCTCCCGGAC92RGTTTTCGTTCGGATCCGCCATTCCTTATCAAGAC93.

[0135] After introducing the vector prepared in this way into B. subtilisKCTC 3135, transformants undergoing single crossover homologous recombination were selected using LB2 agar medium containing chloramphenicol (1% Soytone (Gibco, 212488), 0.5% Yeast extract (Gibco, 212750), 1% Sodium Chloride (Duksan, 82), 1% Agar powder (Duksan, 601)) at 37°C (non-replication temperature). The selected transformants were cultured at 30°C, the replication allowance temperature, and then cultured on antibiotic-free LB agar medium at 39°C to produce colonies undergoing double crossover homologous recombination. Among the generated colonies, those susceptible to chloramphenicol were identified, and plasmid-curing colonies were selected. PCR was performed on the selected colonies using the primers for deletion confirmation in Table 4 under the conditions in Table 2 to confirm whether the target DNA fragment for deletion had been removed from the genome. By sequentially performing the above process and double cross-recombination, a B. subtilisGF#19 strain in which all genes in Table 1 were deleted was produced.

[0136] To confirm whether there is a deletion of the target gene in B. subtilisGF#19, PCR was performed using the deletion confirmation primers of Table 4 on the sequences within the loci of the gene removed in the above experiment, using the genome of B. subtilisGF#19 as a template, and the results of agarose gel electrophoresis are shown in Figure 2.

[0137] Gene locus PCR primer sequence number B. Size of the PCR product of subtilis KCTC 3135 B. Size of PCR product of subtilisGF#19AprEF CAAGGCTTGAAATAACGTTG943115bp2116bpR TTCGCTGATTACAACATTG95NprEF GGAACAGATGATAGCTCATC963487bp2135bpR ACACTCCGAGAAGGCTGAAG97VprF CTGCGGCCTGCACAGCCGCC983733bp2293bpR CGCTGAATGACGGTGGTAAG99NprBF GCTGCTGATTGCAGTAAGCTG1002716bp2153bpR CGCCAATGAAGTGAAGGAGG101MprF GGTTGAGGCGATTGCGACGG1022875bp2130bpR CCATTCTGCAAAATCAGCTC103spoIIAF AAGATATGAAGAAAGCCGGG1042628bp2165bpR ACAGCCGCTTCATAAGCCTG105BprF TCTGCTTTGATGGGTATCGG1065514bp2348bpR AAGCCAAATGTCACCACCAC107EprF ATGATGGACTCCGTTGAGCC1083279bp2469bpR GGCTGGCTTTTTTCTGGTGG109srfACF GGAGCGAAACATATGAGTC1103566bp2840bpR TTGACGGCTTCGCTGTTGAG111ispAF GCTCAGGGTAATGACAGGTG1122544bp2124bpR CGGCGATAATCTTCGAGTTG113epsEF ACATCAGAAGAAATGACGGC1143232bp2487bpR GCACATTTTAGCGAACCAAG115xfpF GCACGAGAATCTATGAGATG1162501bp1991bpR GAAACTGGTACGTCTGCTCC117lytCF GCTCTTAAAGCACAGACTGT1183343bp1852bpR GCAAACTGAACAATTTGTAT119PBSXF GCACGAGAATCTATGAGATG1201896bpRGACACAAGAGAAATTCTTGAAG121sp-βF GTTGCTGTCAGGCTAAGCAA1221879bpR GTGTCAGGCAAGCCATTGTT123cotEF GTACCGATGCGTGTGGAAAAA1242582bp2036bpR GCTCTTCGATTTGATTGACT125skinF GAAGGCGTCACATATCGGAC1261665bpR CGCCGAAATGTCCTAATTGT127sigEF ATGGTATTCGTGACAGCTGG1289012bp3262bpR GTGATGAAACGGCTTAATAA129hlyⅢF GTGTGTGAATGACAACGTCTT1303401bp2759bpR CCATGATCATTGCTCATGTGACT131safAF GACCCGATCTTTTGATCCA1323824bp1916bpR CAGAAACCGCCGATATGC133wprAF CTATTCAGGAGCCAGAGC1344516bp1831bpR CAACCGCAAGTCTTTTATGTC135spoIVAF GAAGCCCTTCTCTATTCG1363363bp1884bpR AGCGGCCCTGTCAATGAA137

[0138] To confirm the EV productivity of the B. subtilisGF#19 strain, the B. subtilisGF#19 strain was inoculated into 10 ml of LB2 medium (1% Soytone (Gibco, 212488), 0.5% Yeast extract (Gibco, 212750), 1% Sodium Chloride (Duksan, 82)) and incubated with stirring at 37 ℃ and 200 rpm for 16 hours to perform a seed culture. The culture solution from the seed culture was inoculated into 25 ml of main culture medium and incubated with stirring at 37 ℃ and 200 rpm for 24 hours to perform the main culture. The composition of the medium for the main culture is shown in Table 5. The prepared medium was sterilized using a 0.22 μm filter. The culture medium was centrifuged at 8,000×g at 4°C for 20 minutes after incubation to remove cell debris, and the supernatant was collected. The concentration of the collected supernatant was analyzed using a Nanoparticle Tracking Assay (NTA; Malvern, NanoSight NS300). The EV concentration measured by NTA corresponds to the density (OD) of the bacterial culture medium that secreted EVs. 600 It was normalized by dividing by ) and is shown in Table 6 below.

[0139] 1 M Potassium phosphate (pH6.5)20ml / LMgSO4·7H2O4g / LNaCl20mg / LNa-citrate (Na3C6H5O7·2H2O)10g / LGlutamic acid monosodium (C5H8NNaO4·H2O)2g / L(NH4)2SO410g / LGlucose50g / LCuSO4· 5H2O20mg / LMnSO4· 7H2O199mg / LFeSO4· 7H2O20mg / LCaCl2· 2H2O80mg / LZnSO4· 7H2O288mg / L

[0140] batch KCTC 3135GF#19#19.4E+087.3E+09#22.4E+091.1E+10#32.1E+092.1E+10 Mean 1.8E+091.3E+10 Standard deviation 7.5E+086.9E+09

[0141] The EV productivity of the B. subtilis GF#19 strain increased by approximately 7.3 times compared to the wild-type strain (KCTC 3135).

[0142] Example 1.2. Preparation of SOD-expressing strain Bs_GF#19-pBE1-SODA2

[0143] The sodA2 gene encoding superoxide dismutase 2 (SOD2) was amplified by duplicate extension PCR using the primers for pBE1-sodA2 preparation in Table 7, using the genome of Bacillus amyloliquefaciens GF423 (accession number: KCTC 13222BP) deposited at the Korea Culture Collection Center (KCTC) as a template. The sodA2 gene sequence amplified by duplicate extension PCR (Sequence No. 3) and the amino acid sequence expressed by it (Sequence No. 4) are presented in Table 8 (Genetic sequence introduced into Bs_GF#19-pBE1-SODA2).

[0144] Primer sequence (5' → 3') SEQ ID NO: 1SOD FGGAGGAATTACAATGGCTTACAA1382D74 RTGCATGTCCGCCGCCATCGTTGCGGAC1393D74 FCAGTCCGCAACGATGGCGGCGGACATG1404D137 RTTCAAGTTTGCCGTCGTTTACAACGAGC1415D137 FCTCGTTGTAAACGACGGCAAACTTG1426SOD RCAAAACAGAAGCTTCATTATTTTGCT143

[0145] Name Sequence Sequence Number sodA Nucleotide sequence of the gene Amino acid sequence of the gene MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNNGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNNGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK2sodA2 Nucleotide of the gene서열ATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACGATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACGACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAATAA3sodA2 유전자의 아미노산 서열MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNDGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNDGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK4

[0146] The construct generated by assembling an amplified DNA fragment containing the sodA2 gene and the plasmid pBE1, linearized by treatment with NdeI and HindIII, in vitro using the SLIC method (Applied and Environmental Microbiology. 2012.78(15):5440-5443) was introduced into E. coli C2984H for transformation. Clones into which the plasmid was introduced were screened by restriction enzyme mapping, and finally, nucleotide sequencing was used to confirm the introduction of the gene into the plasmid and the E. coli C2984H clones transformed with the corresponding plasmid. The expression vector pBE1-sodA2 constructed through this process is shown in Fig. 3. pBE1-sodA2 was introduced into B. subtilisGF#19 to transform B. subtilisGF#19. The gene-introduced strains from the transformants were selected via polymerase chain reaction using primers for the plasmid region and specific primers for the inserted gene region, and pure clones were established by two single-colony isolation procedures in LB2 (1% Soytone, 0.5% Yeast extract, 1% Sodium Chloride) medium. The selected strain was named Bs_GF#19-pBE1-SODA2 and stored at -80 ℃ using the glycerol stock method.

[0147] Example 1.3. Preparation of Blue Fluorescent Protein-SOD Fusion Protein (SODA2-EBFP) Expressing Strain, Bs_GF#19-pBE1-SODA2-EBFP

[0148] To label SODA2 with blue fluorescence, enhanced blue fluorescent protein (EBFP) was fused to the C-terminus of SODA2. The gene encoding EBFP was synthesized by Bionia Inc. (Daejeon, South Korea), and its nucleotide sequence is as shown in Sequence No. 5 in Table 10. Using the synthesized gene of Sequence No. 5 as a template, the DNA fragment amplified by PCR using the primers for pBE1-sodA2-EBFP preparation in Table 9 and the plasmid pBE1, which was linearized by treatment with restriction enzymes NdeI and HindIII, were assembled in vitro in the same manner as in Example 1.2. The resulting construct was then introduced into E. coli C2984H and transformed in the same manner as in Example 1.2. Clones with the introduced gene were selected by restriction enzyme mapping and confirmed by nucleotide sequencing. A schematic diagram of the prepared plasmid, pBE1-sodA2-EBFP, is shown in Fig. 4. The expression vector pBE1-sodA2-EBFP was introduced into B. subtilisGF#19 to transform B. subtilisGF#19. It was selected and stored in the same manner as in Example 1.2.

[0149] Primer sequence (5' → 3') Sequence No. 1 SOD FGGAGGAATTACAATGGCTTACAA1382sodA2 R(linker)CCACCTCCGCCTCCTTTTGCTTCGCTGTAAAGA1443EBFP F(linker)GGAGGCGGAGGTGGAGTGTCGAAAGGAGAAGAAT1454EBFP RCAAAACAGCCAAGCTTGTTATTTATACAGTTCA146

[0150] Name Sequence Sequence Number Amino acid sequence of blue fluorescent protein MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTHGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK5

[0151] Example 1.4. Preparation of a DNA fragment containing the blue fluorescent protein (EBFP) gene

[0152] To verify whether genes delivered via EVs undergo transcription and translation in mitochondria, DNA fragments fused with the EBFP gene to a mitochondrial promoter were prepared. Specifically, to extract mitochondrial DNA, human retinal pigment epithelial cells APRE19 (ATCC, CRL2302) were cultured in DMEM F12 (1:1) medium supplemented with 10% FBS and 1% penicillin / streptomycin (Gibco, 11320-033) at 37°C and 5% CO2. Subsequently, the APRE19 cells were detached using 0.5% Trypsin-EDTA mixed with 1×dPBS in a 1:1 ratio (Gibco, 15400-054) and centrifuged at 3,000×g for 3 minutes to obtain 1.0×10⁻⁶ 7 Number of cells were obtained. The obtained cell pellet was suspended in RIPA (Thermo, 89901) buffer containing a protease inhibitor cocktail (Roche, 05056489001), and then Exprep TM Mitochondrial DNA was extracted using the plasmid SV kit (Geneall, 101-102).

[0153] To obtain an EBFP DNA fragment linked to an intramitochondrial expression promoter, the extracted mitochondrial DNA was used as a template, and PCR was performed using the primers for preparing the EBFP DNA fragment linked to the intramitochondrial expression promoter in Table 11 to obtain a DNA fragment containing the intramitochondrial expression promoter. The DNA fragment containing the intramitochondrial expression promoter was assembled in vitro with the EBFP gene fragment using the SLIC method. A schematic diagram of the prepared DNA fragment, mt_P2-EBFP, is shown in Fig. 5.

[0154] Primer sequence (5' → 3') Sequence number 1 mt-FGGAGGAATTACAGATCACAGGTCTATCACCCT1472 mt-RGTTTTAAGTTTTATGTTGTTTATGGGGTGATGTGAGCCCGTCT1483 tR(1 / 2) FCACCCCATAAACAACATAAAACTTAAAACTTTACAGTCAGAGGTT1494 tR(1 / 2) RCTTTCGACACCATGTTGTTAAGAAGAGGAATTGA1505EBFP(tR) FCTTCTTAACAACATGGTGTCGAAAGGAGAAGAAT1516EBFP RCAAAACAGCCAAGCTTGTTATTTATACAGTTCA146

[0155] Example 2.B. Preparation of subtilis-derived EV

[0156] Example 2.1.B. subtilis culture

[0157] The B. subtilis GF#19 strain was inoculated into 200 ml of LB2 medium (1% Soytone (Gibco, 212488), 0.5% Yeast extract (Gibco, 212750), 1% Sodium Chloride (Duksan, 82)) and incubated with stirring at 37 ℃ and 200 rpm for 16 hours to perform a seed culture. The culture medium from the seed culture was inoculated into 200 ml of main culture medium and incubated with stirring at 37 ℃ and 200 rpm for 24 hours to perform the main culture. The composition of the medium for the main culture is shown in Table 5. The prepared medium was sterilized using a 0.22 μm filter.

[0158] Example 2.2.B. Separation and Purification of EV Derived from Subtilis

[0159] The culture medium was centrifuged at 8,000×g at 4°C for 20 minutes to remove cell debris, and the supernatant was recovered. The recovered supernatant was filtered using a 0.2 μm filter via a Tangential Flow Filtration (TFF) system. Substances smaller than the filter's Molecular Weight Cut Off (MWCO) were removed using a 750 kDa hollow fiber filter (Repligen, D02-E750-05-N), and then the solution was concentrated to 1 / 10 of its volume to finally isolate EVs. The isolated EVs were filtered and sterilized using a 0.22 μm syringe filter, and their particle size and concentration were analyzed using a Nanoparticle Tracking Assay (NTA; Malvern, NanoSight NS300). The average size of Bs_GF#19EV is 82.7 nm, and the concentration is 5.42×10 11 It was particle / mL (Fig. 6a).

[0160] Example 2.3. Manufacture of SODA2-equipped B. subtilisEV

[0161] EVs were prepared from the Bs_GF#19-pBE1-SODA2 strain using the same method as in Examples 2.1 and 2.2, and their particle size and concentration were analyzed. The EVs produced from the Bs_GF#19-pBE1-SODA2 strain had an average size of 78.0 nm and a value of 3.12 × 10⁻⁶ 10 The concentration value of particle / mL was checked (Fig. 6b).

[0162] Example 2.4. Preparation of an EV loaded with a blue fluorescent protein (EBFP)-SOD fusion protein

[0163] EVs were prepared from the Bs_GF#19-pBE1-SODA2-EBFP strain using the same method as in Examples 2.1 and 2.2, and their particle size and concentration were analyzed. The EVs produced from Bs_GF#19-pBE1-SODA2-EBFP had an average size of 72.3 nm and 7.74×10⁻⁶ 10 The concentration value of particle / mL was checked (Fig. 6c).

[0164] Example 2.5. Preparation of an EV loaded with a DNA fragment containing the blue fluorescent protein (EBFP) gene

[0165] An mt_P2-EBFP DNA fragment was loaded onto an EV prepared from the B. subtilisGF#19 strain by the same method as in Examples 2.1 and 2.2 using an electroporation method. Specifically, an mt_p2-EBFP DNA fragment (5 uL) was loaded with 5×10 11195 μL of EVs produced from B. subtilis GF#19 at a particle / mL concentration were mixed and left on ice for 30 minutes. Subsequently, the mixture was mixed 1:1 with 400 mM sucrose diluted with distilled water, transferred to a 2 mm electroporation cuvette (Cell Projects, EP-202), and subjected to electroporation at 2.5 kV and 50 μF. After electroporation, the samples were incubated in a 37 ℃ incubator for 30 minutes without shaking; particle size and concentration were analyzed using an NTA (Malvern, Nanosight NS300) instrument for experimental use. The EVs electroporated from mt-EBFP had an average size of 87.1 nm and a concentration of 1.84 × 10⁻⁶ 11 The concentration was particles / mL (Fig. 7a), and the electroporated EVs of mt_p2-EBFP had a size of 84 nm and 1.73×10 11 It had a concentration of particles / mL (Fig. 7b).

[0166] Example 3. Mitochondrial selective delivery of protein using Bacillus-derived EV

[0167] Example 3.1. Mitochondrial selective delivery of blue fluorescent protein (EBFP)-SODA2 fusion protein

[0168] To investigate whether EVs purified from *B. subtilis* can selectively deliver proteins to mitochondria, Vero cells (Korean Cell Line Bank, KCLB No. 10081), which are African green monkey kidney cells, and GM13411 human fibroblast cells (Coriell Institute, GM13411) were treated with EVs loaded with SODA2-EBFP (SODA2 labeled with blue fluorescent protein), prepared as in Example 2.4, and the intracellular localization of blue fluorescence was observed. Mitochondria were stained using a MitoTracker (Thermo Fisher Scientific, M7512). The specific test methods are as follows.

[0169] Vero cells were inoculated into Tomodish (Tomocube, Tomodish#1.5H) at a confluency of 20–40% using MEM (Welgene, LM 007-07) supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. The cells were cultured for 3 hours after treatment with EVs loaded with EBFP-SODA2 fusion proteins. To confirm whether the EBFP-SODA2 fusion protein was delivered to mitochondria, the cells were treated with MitoSox for 20 minutes and 100 nM Mitotracker for 30 minutes after EV treatment. The delivery of the EBFP-SOD fusion protein introduced into the cells to mitochondria was analyzed using HT-2T (tomocube, HT-2H).

[0170] GM13411 cells were also inoculated into Tomodis at 20–40% confluence using MEM supplemented with 15% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2 conditions, just like Vero cells. The assay to confirm whether the EBFP-SODA2 fusion protein is delivered to the mitochondria is the same as the assay for Vero cells.

[0171] As a result of the test, purple fluorescence was observed in both Vero cells (Fig. 8a) and GM13411 cells (Fig. 8b) treated with EVs loaded with the EBFP-SODA2 fusion protein, due to the co-localization of blue and red fluorescence. This result is because SODA2 labeled with blue fluorescence was delivered to the mitochondria labeled with red fluorescence. Additionally, since blue fluorescence was not observed in other endoplasmic reticulum or cytoplasm within the cell, it appears that the EBFP-SODA2 fusion protein was selectively delivered to the mitochondria.

[0172] Example 3.2. Effects of mitochondrial delivery of SODA2

[0173] The results of Example 3.1 demonstrated that B. subtilis EVs can selectively deliver proteins to mitochondria. To determine whether proteins delivered to mitochondria could affect mitochondrial function, Vero cells were treated with H2O2 to increase the concentration of mitochondrial superoxide. Subsequently, B. subtilis EVs loaded with SODA2, which was not fused with EBFP, were treated. Changes in mitochondrial superoxide concentration were analyzed using MitoSox (Invitrogen, M36008), a staining reagent capable of specifically staining mitochondrial superoxide. Specifically, Vero cells were inoculated into Tomodici at 20–40% confluence using MEM supplemented with 10% FBS and 1% penicillin / streptomycin under conditions of 37°C and 5% CO2. Cells were treated with a mixture of 50 µM H2O2 and 0.1 mU glucose oxidase and cultured for 3 hours. After culture, 1.0 × 10⁶ 8EVs extracted from Bs_GF#19-pBE1-SODA2 recombinant B. subtilis were treated for 1 hour. To confirm changes in mitochondrial superoxide concentration, red fluorescence was detected using HT-2H after treatment with 5 µM MitoSox for 20 minutes. Fluorescence intensity was quantified using ImageJ, and statistical analysis was performed using multiple comparisons (Dunnett's test) in Prism software.

[0174] Referring to Figure 9, treatment of Vero cells with H2O2 increased red fluorescence within the mitochondria, indicating an increase in the concentration of mitochondrial superoxide. When treated with EVs without SODA2, there was no change in fluorescence within the mitochondria compared to the H2O2-treated group. On the other hand, when treated with EVs loaded with SODA2, red fluorescence decreased statistically significantly. These results are due to the removal of superoxide by SODA2 delivered to the mitochondria via recombinant B. subtilis-derived EVs.

[0175] Mitochondrial membrane potential is an indicator of mitochondrial function and health. To investigate whether delivered SODA2 could affect mitochondrial membrane potential, the state of mitochondrial membrane potential was observed using the JC1 (Invitrogen, T3168) staining reagent, which is capable of measuring membrane potential.

[0176] Specifically, HeLa cells (Korean Cell Line Bank, KCLB No. 10002), a cervical cancer cell line, were inoculated into Tomodici with 20–40% Confluence using DMEM (Welgene, LM 001-05) supplemented with 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. The cells were treated with a mixture of 50 µM H2O2 and 0.1 mU glucose oxidase and cultured for 3 hours. After culture, the cells were washed three times with 1×dPBS, followed by 1.0×10 8 EVs extracted from Bs_GF#19-pBE1-SODA2 recombinant B. subtilis were treated for 1 hour. To check the membrane potential, red and blue fluorescence were measured using HT-2H after treatment with 7 uM JC1 for 20 minutes (Fig. 10a). Fluorescence intensity was quantified using ImageJ, and the results are shown in Fig. 10b. The JC1 ratio is the ratio of green fluorescence intensity to red fluorescence intensity. Statistical analysis was performed using the multiple comparison (Dunnett's test) of Prism software.

[0177] In the group of HeLa cells treated only with H2O2, staining of mitochondria with JC1 revealed a decrease in the ratio of red fluorescence to green fluorescence, confirming that the mitochondrial membrane potential had depolarized. Conversely, in the group treated with H2O2 followed by an EV loaded with SODA2, the ratio of red fluorescence increased, and the JC1 ratio also increased statistically significantly compared to the group not treated with the SODA2-loaded EV.

[0178] These results are due to the fact that SODA2 within the EVs of recombinant B. subtilis was delivered to the mitochondria, and the integrity of the mitochondrial membrane was restored by removing superoxide.

[0179] Example 4.B. Mitochondrial selective delivery of nucleic acids using subtilis-derived EVs

[0180] Example 4.1. Delivery of the blue fluorescent protein gene

[0181] To determine whether the EV of B. subtilis could deliver DNA to the mitochondria, a DNA fragment (mt_p2-EBFP) was prepared by fusing the EBFP gene with a mitochondrial promoter so that it could be transcribed by the mitochondrial promoter as in Example 2.5, and this DNA fragment was loaded onto the EV by electroporation. Whether the DNA fragment loaded onto the EV could be delivered to the mitochondria and transcribed and translated was analyzed by observing blue fluorescence within the mitochondria.

[0182] Specifically, GM13411 cells were inoculated into Tomodici at 20–40% Confluence using MEM supplemented with 15% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2. The cells were treated with EVs loaded with mt_p2-EBFP via electroporation. After EV treatment, the cells were treated with 100 nM Mitotracker for 30 minutes to confirm whether mt_p2-EBFP was delivered into the mitochondria. The expression of EBFP induced by mt_p2-EBFP within the mitochondria was confirmed using HT-2H.

[0183] Referring to Figure 11, blue fluorescence is observed in the group treated with EVs loaded with mt_p2-EBFP, and since the blue fluorescence is observed at the same location as the fluorescence of the Mitotracker, it indicates that the EBFP gene was transcribed and translated within the mitochondria. This means that when cells are treated with EVs of recombinant B. subtilis, DNA fragments within the EVs are delivered into the mitochondria and successfully transcribed and translated.

[0184] Example 5. Intracellular influx of Gram-positive bacteria other than B. subtilis

[0185] Example 5.1 Culture of microorganisms

[0186] 200 ml of Difco TM Seed culture was performed by inoculating into Lactobicilli MRS Broth (Difco, 288130) medium and incubating at 37°C for 16 hours. The prepared medium was sterilized by autoclaving. The main culture solution was inoculated into 200 ml of new medium with the same composition as the existing medium and main culture was performed by incubating at 37°C for 24 hours.

[0187] Bacillus velezensis FZB42 strain (accession number: KCTC 1660) and Bacillus velezensis GF423 strain (accession number: KCTC 13222BP), deposited at the Korea Culture Collection Center (KCTC), were inoculated into 10 ml of LB2 medium (1% Soytone (Gibco, 212488), 0.5% Yeast extract (Gibco, 212750), 1% Sodium Chloride (Duksan, 82)) and incubated with stirring at 37°C and 200 rpm for 8 hours to perform seed culture. The culture medium was inoculated into 25 ml of new medium with the same composition as the existing medium and incubated with stirring at 37°C and 200 rpm for 16 hours to perform the main culture. The prepared medium was sterilized using a 0.22 μm filter.

[0188] Example 5.2. Isolation and Purification of Microbial-Derived EV

[0189] EVs derived from L. paracasei were isolated and purified in the same manner as in Example 2.2, with an average EV size of 79.4 nm and a concentration of 6.38 × 10⁻⁶ 10 It was particle / mL (Fig. 12a).

[0190] For EVs derived from *B. velezensis*, the culture medium was centrifuged at 8,000×g at 4°C for 20 minutes to remove cell debris, and the supernatant was recovered. The recovered supernatant was filtered using a syringe filter with a pore size of 0.2 μm. Substances smaller than the filter's molecular weight cut-off (MWCO) were removed using a 100 kDa Vivaspin Turbo 15 (SATORIUS, VS15T42), and the solution was concentrated to 1 / 10 of its volume to finally isolate the EVs. The isolated EVs were filtered and sterilized using a 0.22 μm syringe filter, and their particle size and concentration were analyzed using a Nanoparticle Tracking Assay (NTA; Malvern, NanoSight NS300). The average size of *B. velezensis* FZB42 EVs was 99.3 nm, and the concentration was 8.47×10⁻⁶. 09 It was particle / mL (Fig. 12b), the average size of B. velezenesis GF423 EV was 113.9 nm, and the concentration was 3.95 × 10⁻⁶ 11 It was particle / mL (Fig. 12c).

[0191] Example 5.3 Preparation of EV Labeled with Lipid-Soluble Fluorescent Dye (DiO)

[0192] A lipophilic fluorescent dye was labeled onto EVs prepared from Gram-positive bacterial strains using the same method as in Example 5.2. Specifically, the NaCl concentration of the EV suspension was adjusted to be 20 mM or less, and then reacted with 2 μM DiO₂; DiOC18 (Invitrogen, D275) dye at 37°C for 30 minutes. After the reaction, a small amount of 10X PBS was added to adjust the NaCl concentration to approximately 150 mM. Subsequently, the reaction product was filtered and sterilized using a 0.22 μm syringe filter, and the particle size and concentration were analyzed using a Nanoparticle Tracking Assay (NTA; Malvern, NanoSight NS300) instrument to determine the value of 1×10⁻⁶. 8 It was used for endocytosis at particle / mL.

[0193] Example 5.4 Intracellularization of EVs derived from Gram-positive bacteria

[0194] To confirm whether EVs derived from Gram-positive bacteria are endocytosed, eukaryotic cells were treated with a lipid-soluble fluorescent dye as in Example 5.3. Specifically, Vero cells were inoculated into Tomodisi at 20–40% Confluence using MEM supplemented with 10% FBS and 1% penicillin / streptomycin under conditions of 37°C and 5% CO2. Subsequently, the cells were treated with EVs labeled with a lipid-soluble fluorescent dye for 3 hours. After EV treatment, the endocytosed of EVs labeled with a lipid-soluble fluorescent dye was confirmed by HT-2H analysis (Fig. 13).

Claims

1. A composition comprising extracellular vesicles derived from Gram-positive bacteria for the selective delivery of an active ingredient to mitochondria.

2. In Paragraph 1, The above Gram-positive bacteria belong to the genus Lacticaseibacillus or Bacillus. Composition.

3. In Paragraph 1, The above Gram-positive bacteria belong to the Bacillus subtilis species complex. Composition.

4. In Paragraph 1, The above Gram-positive bacteria are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Lactobacillus paracasei. Composition.

5. In Paragraph 1, The above composition further comprises an active ingredient that needs to be delivered to mitochondria. Composition.

6. In Paragraph 1, The above active ingredient exists in a form that forms a complex with the extracellular vesicle or is contained within it. Composition.

7. In Paragraph 1, The above extracellular vesicles can penetrate the cell membrane and selectively move into mitochondria. Composition.

8. In Paragraph 1, The extracellular vesicles derived from the Gram-positive bacteria have an average particle size in the range of 1 nm to 300 nm. Composition.

9. In Paragraph 5, The above active ingredient is a polypeptide, nucleic acid, or a combination thereof. Composition.

10. In Paragraph 9, The above active ingredient is an ingredient for preventing, improving, or supplementing mitochondrial dysfunction or cellular energy deficiency. Composition.

11. In Paragraph 9, The above active ingredient is superoxide dismutase (SOD) or a nucleic acid encoding it. Composition.

12. In Paragraph 1, The above active ingredient is superoxide dismutase derived from Bacillus velezensis. Composition.

13. In Paragraph 1, The above composition is a food or cosmetic composition. Composition.

14. In Paragraph 1, The above composition is a pharmaceutical composition for the prevention or treatment of cellular energy deficiency or mitochondrial dysfunction. Composition.

15. A method for preparing a composition of any one of claims 1 to 14, comprising the steps of culturing a Gram-positive bacterial strain and isolating extracellular vesicles from the culture, The above-mentioned Gram-positive bacterial strain is genetically modified to produce an active ingredient that needs to be delivered to the mitochondria within the cell, or The method further comprises the step of mixing the above-described isolated extracellular vesicles derived from Gram-positive bacteria with an active ingredient that needs to be delivered into intracellular mitochondria. method.

16. In Paragraph 15, Delivery to intracellular mitochondria is performed in vitro, in vitro, or in vivo. method.

17. A recombinant Gram-positive bacterial strain with improved extracellular vesicle production ability compared to unmodified Gram-positive bacteria.

18. In Paragraph 17, The above strain is a strain belonging to the genus Lacticase Bacillus or Bacillus. Recombinant Gram-positive bacterial strain.

19. In Paragraph 17, The above strain is a strain belonging to the Bacillus subtilis species complex. Recombinant Gram-positive bacterial strain.

20. In Paragraph 17, The above strain is a Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Lacticasei Bacillus paracasei strain. Recombinant Gram-positive bacterial strain.

21. In Paragraph 17, The above strain is one in which one or more genes selected from the group consisting of AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAC, EpsE, Xpf, lytC, PBSX, SP-β, cotE, skin, sigE, hlyⅢ, wprA, Saf-coxA, and spoIVA are deleted compared to the wild-type strain. Recombinant Gram-positive bacterial strain.

22. In Paragraph 21, The above strain is one in which one or more genes selected from the group consisting of AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, EpsE, and wprA are deleted compared to the wild-type strain. Recombinant Gram-positive bacterial strain.

23. In Paragraph 17, The above-mentioned recombinant Gram-positive bacterial strain further includes a foreign gene encoding an active substance for delivery to mitochondria. Recombinant Gram-positive bacterial strain.

24. A step of administering a composition according to any one of claims 1 to 14 to a subject A method for delivering an active substance to mitochondria.

25. A step of administering a composition according to any one of claims 1 to 14 to a subject A method for preventing, improving, or treating cellular energy deficiency or mitochondrial dysfunction.

26. In Paragraph 24, The above mitochondrial dysfunction or cellular energy deficiency is selected from the group consisting of Leigh Syndrome, MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes), Leber Hereditary Optic Neuropathy (LHON), Kearns-Sayre Syndrome (KSS), MERRF (Myoclonic Epilepsy with Ragged-Red Fibers), and Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP). method.

27. Use of a composition according to any one of claims 1 to 14 for preventing, improving, or treating cellular energy deficiency or mitochondrial dysfunction.

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