KR20180036134A - Process for preparing exosome loading super-repressor-IκB protein, and pharmaceutical composition for use in preventing or treating inflammatory diseases containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a method for mass-producing an exosome containing a super-repressor-IκB (EXPLOR) protein, a vector for producing an exosome which can be used for producing the exosome, the exosome which is produced by the method and contains the super-repressor-IκB protein, and a pharmaceutical composition for preventing and treating inflammatory diseases containing the same as an active ingredient. By using the method for producing the exosome containing the super-repressor-IκB protein of the present invention, it is possible to produce the exosome containing the super-repressor-IκB protein with high yield. Accordingly, the exosome can be widely used for treating inflammatory diseases.

Description

TECHNICAL FIELD The present invention relates to a process for preparing an exosome containing super-repressor-IκB protein and a pharmaceutical composition for the prevention and treatment of an inflammatory disease containing an exosome prepared by the above- protein, and pharmaceutical composition for use in preventing or treating inflammatory diseases comprising the same as an active ingredient}

The present invention relates to a method for producing an exosome containing a super-repressor-IκB protein using a photo-specific binding protein and a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases containing the exosome prepared by the above- ≪ / RTI >

The human body is composed of about 200 kinds of 100 trillion cells, and its bioactivity is regulated by the action of various proteins in the cell to maintain its life.

Cells are surrounded by a bilayer membranous membrane composed of phospholipids, which blocks the entry of foreign substances into cells. Most of the protein therapeutics developed so far also pass through the cell membrane and can not enter the cytoplasm. They have physiological effects by acting outside the cell or by acting on a receptor on the cell membrane to transmit signals into the cell.

There are numerous proteins in the cytoplasm, which interact with each other to regulate their physiological activity. If the protein therapeutic can be put directly into the cell, that is, into the cytoplasm, the cell activity will be controlled more effectively.

Recently, studies on the direct introduction of the target protein into the cell through the cell membrane have been actively conducted. Protein Transduction Domains (PTDs), which are peptides that pass through the cell membrane, and recombinant proteins of the target protein can be prepared and introduced into the cytoplasm through the cell membrane (FIG. 1). The PTDs include HIV-1 TAT, HSV VP22, Antp, dfTAT, and Hph-1. Fusion proteins that bind these proteins with target proteins need to be produced and separated in the form of recombinant proteins. There is a problem in that refolding of the protein is not performed properly, the activity is decreased, the protein is nonspecifically transmitted, the risk of causing an immune reaction in vivo is large, the cost is high, and the yield is low.

The target protein bound to various nanoparticles can enter the cytoplasm through the cell membrane by endocytosis (Fig. 2). The nanoparticles include Gold NP, Liposome NP, Magnetic NP, and Polymeric NP. Most of the degradation products of these proteins and proteins bind to lysosomes in the cell, , It is difficult for the target protein and the nanoparticles to separate from each other in the cytoplasm, and the toxicity of the nanoparticles may become a problem.

Exosome refers to a small vesicle of membrane structure with a size of 50 - 200 nm that is secreted out of the cell with protein, DNA, RNA, etc., for intercellular signaling.

Exosomes were originally found in the process of leaving hemoglobin in the red blood cells by eliminating and exiting the intracellular proteins at the last stage when the red blood cells were matured. These exosomes were found in the electron microscopic study, But rather from a specific compartment within the cell called Multivesicular bodies (MVBs), which is released and released outside the cell. That is, when fusions between the polycation and the plasma membrane occur, such follicles are released into the extracellular environment, which is called exosomes (FIG. 3).

Although it is unclear how these exosomes are produced by any molecular mechanism, it is possible to identify a variety of immune cells and stem cells, including B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, Tumor cells are also known to produce and secrete exosomes in a living state.

Exosomes contain various proteins, DNA, and RNA in the cell. The substances contained in the exosome and secreted out of the cell may be re-introduced into other cells by fusion or endocytosis with the cell membrane and serve as intercellular communication partners. In addition, the substances contained in the exosome and secreted out of the cell can be analyzed and used for diagnosis of a specific disease.

Several types of microRNAs are contained in exosomes, and a method for diagnosing disease by detecting the presence or abundance thereof is known (see KR 10-2010-0127768A). In particular, WO2009-015357A discloses a method for detecting exosomes present in a cancer-derived sample (blood, saliva, tears, etc.) and measuring the amount of microRNA change. When the increase or decrease is compared with the control, And a method for diagnosing the abnormality is disclosed. Particularly, exosomes obtained from patients having a specific disease (lung disease) are analyzed to specifically describe the relationship between a specific microRNA and lung disease. Also, in addition to pulmonary disease, methods for diagnosing renal disease using protein contained in exosome have been studied.

Exosomes also contain antigens. On the other hand, in antigen presenting cells (APCs), antigen peptides are loaded on MHC (major histocompatibility complex) class II molecules in intracellular compartments containing membranes, including polycations. Therefore, Peptide-MHC class II complex. Thus, exosomes can present antigen peptides to CD4 + T lymphocytes as transporters of immunogens, thereby inducing an immune response such as proliferation of T lymphocytes. In addition, since exosomes are enriched with molecules capable of stimulating an immune response, such as MHC class I, heat-shock proteins (HSPs), they are immune to autoimmune disease or tumor treatment May be used for the purpose of enhancing or reducing.

In addition, the exosome in which the target protein is contained can be used for the treatment of various diseases in vivo. For example, a protein exhibiting an anti-cancer effect as a target protein, or an exosome containing siRNA can be prepared, and then treated with cancer cells to treat cancer (FIG. 4).

Thus, exosomes containing the desired protein can be used for the treatment of disease. For this purpose, it is necessary to be able to efficiently produce exosome containing the target protein. Korean Patent Publication No. 2004-0015508 describes a method for producing an exosome comprising a specific antigen. There is disclosed a method in which a gene for a specific antigen is introduced into a cell line and the protein of the introduced gene is stably expressed in the cell line and is released to the outside of the cell through the exosome and a method of using such exosome as a vaccine .

However, since the exosome is formed naturally in the cell, even if a gene encoding the target protein is introduced into the cell that produces the exosome, the exosome in which the expressed protein is contained can be produced Is very low.

Accordingly, the present inventors have made efforts to develop a method for more effectively producing an exosome containing a target protein. As a result, they have found that a fusion protein obtained by fusing an exosome-specific marker and a target protein is expressed in a cell producing a large amount of exosome , It was confirmed that the exosome containing the target protein can be efficiently produced (Fig. 5).

In addition, in the above method, the target protein is attached to the membrane of the exosome, and a fusion protein obtained by fusing each of the light-specific binding protein pairs to each of the exosome-specific marker and the target protein is called a cell that produces a large amount of exosome When these fusion proteins are introduced into the exosome by the exosome-specific marker and the irradiation of the light is stopped after the introduction of the exosome-specific marker, the exosome- It was confirmed that the fusion protein of the red binding protein was isolated and the exosome containing the target protein in the free state could be effectively produced (FIG. 6).

Although the inflammatory response is a body defense mechanism for a variety of stimuli (Anderson et al., Pharmacol. Rev. 60: 311357, 2008), chronic inflammatory reactions are associated with various diseases including arthritis, hepatitis, septic shock and neuropathic disorders (Chung et al., Clin. Rheumatol. 26: 12281233, 2007). LPS has been implicated in the production of nitric oxide (NO), prostaglandin E2 (PGE2), and the like, as well as in a variety of inflammatory responses (Mayeux et al., J. Toxicol. Activates macrophages that produce inflammatory mediators and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) (Takeuchi et al., J. Biol. Chem. 281: 2136221368 , 2006). Inducible nitric oxide synthase (iNOS) promotes the formation of excessive nitric oxide that can evolve into inflammatory diseases (Nathan et al., Curr. Opin. Immunol. 3: 6570, 1991), cyclooxygenase- (COX-2) is induced by inflammatory stimulation and reaction for the synthesis of PGE2 (Yoon et al., J. Biosci. Bioeng. 107: 429438, 2009). Thus, overexpression of NO and PGE2 by iNOS and COX-2 plays an important role in regulating the inflammatory response.

NF-κB is a major transcription factor that induces inflammatory responses, mainly regulating the expression of inflammation-related genes in immune cells and various other cells. Thus, excessive activity of the NF-κB signaling pathway leads to a variety of inflammatory diseases, thus selectively inhibiting the over-activated NF-κB pathway in inflammatory cells is an effective treatment strategy for refractory chronic inflammatory diseases such as rheumatoid arthritis, . In addition, since activation of NF-κB increases the expression of factors capable of inhibiting apoptosis and thus inhibits apoptosis, the activation of the NF-κB signal pathway in cancer cells is a major cause of tolerance to chemotherapy And thus the therapeutic effect of the anticancer agent is reduced.

In unstimulated normal cells, most NF-κB binds to its inhibitory protein, IκB, and remains inactive in the cytoplasm. IκB kinase (IKK) complexes activated by various stimuli such as TNF-α and LPS phosphorylate IκB, and phosphorylated IκB is ubiquitinated, resulting in degradation through proteasomes. As IκB is degraded, the NF-κB (p50 / p65) bound to it separates from the cytoplasm in a free state, passes through the nuclear membrane, and then binds to the promoter portion of the target gene in the nucleus to promote mRNA transcription (Lappas et al., Biol. Reprod. 67: 668673, 2002), which induces the transcription of inflammatory mediators and inflammatory cytokines such as iNOS, COX-2, NO, PGE2, TNF-α and IL- Is an important element of.

Super-repressor IκB is a S32A, S36A mutant form of IκB that is not phosphorylated by IKK and is not degraded by the proteolytic enzyme complex, resulting in the ability to continuously inhibit NF-κB. Thus, there is a very high possibility as a therapeutic agent in various inflammatory disease models.

Accordingly, the present inventors prepared exosome containing the super-repressor IκB protein and confirmed that the super-repressor IκB protein was transferred to the cytoplasm of the target cell when the exosome was treated, and the super-repressor IκB of the present invention Confirming that the loaded exosome can be used as an agent for treating inflammatory diseases.

It is an object of the present invention to provide a method for mass-production of an exosome comprising a fusion protein of a protein that inhibits exosome-specific markers and NF-κB.

Another object of the present invention is to provide a method for mass-production of an exosome comprising a protein which inhibits NF-κB isolated from the exosome membrane using a photo-specific binding protein pair.

Another object of the present invention is to provide a vector for producing an exosome which can be used for the production of the exosome.

It is another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the exosome as an active ingredient.

According to an aspect of the present invention,

a) obtaining a transformed cell by introducing into the exosome-producing cell a polynucleotide encoding a fusion protein in which a protein that inhibits exosome-specific markers and NF-κB is bound; And b) culturing the transformed cells. The present invention provides a method for mass-producing exosome to which an NF-κB inhibiting protein is attached to an exosomal membrane.

In addition,

a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide that binds to the first photo- Introducing a polynucleotide encoding a fusion protein (second fusion protein) in which a second photo-specific binding protein capable of binding to NF-κB is bound to a protein that inhibits NF-κB; b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And c) stopping the irradiation of the light after the exosome is produced in the exosome-producing cell. The present invention also provides a method for mass-producing exosome comprising a protein inhibiting NF-κB.

In addition,

(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first light-specific binding protein and an exosome-specific marker are bound; And (b) a polynucleotide capable of introducing a polynucleotide encoding a protein that inhibits NF-κB and a polynucleotide encoding a second photo-specific binding protein capable of binding the first photo- And a second expression vector comprising the second expression vector.

In addition, the present invention provides a method for delivering a protein inhibiting NF-κB to the cytoplasm, which is produced using the above method, wherein the exosome containing the protein inhibiting NF-κB is used, and the exosome is used And a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases comprising the exosome as an active ingredient.

The present invention provides a method for producing an exosome comprising an NF-κB inhibiting protein, which can produce an exosome containing a protein that inhibits NF-κB at a high yield, Can be widely used for the treatment of inflammatory diseases by using the exosome.

FIG. 1 is a diagram showing a cytoplasmic delivery method of a target protein through a cell membrane transduction domain (PTD) and a recombinant protein of a target protein (Steven R. et al., Protein transduction: unrestricted delivery into all cells, Trends in Cell Biology, .
FIG. 2 is a graph showing the intracellular delivery of a target protein through endocytosis to a conjugate comprising nanoparticles and a target protein (Munish Chanana et al., Physicochemical properties of protein-coated gold nanoparticles in biological fluids and cells before and after proteolytic digestion, Angew, Chem., Int.
FIG. 3 is a view showing the process in which exosomes are released from the cells and released from the cells of the multi-vesicular bodies (MVBs) (Grace Raposo and Willem Stoorvogel, Extracellular vesicles: Exosomes, microvesicles, and friends. (4), 373-383, 2013).
FIG. 4 is a diagram showing a process of delivering siRNA in vivo through a targeted exosome to treat cancer (Alvarez-Erviti, L. et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature biotechnology 29, 341-345, 2011).
FIG. 5 is a schematic diagram showing a process for producing protein-carrying exosomes (EXPLORs) designed as optogenetically-designed proteins according to the present invention.
FIG. 6 is a diagram showing a process of separating the fusion protein of the target protein and the light-specific binding protein from the inside of the exosome when the light irradiation of EXPLORs according to the present invention is stopped.
FIG. 7 is a fluorescence photograph showing the change in the intracellular location of mCherry protein in a transformant into which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells according to irradiation of blue light.
8 is a diagram showing an experimental procedure for obtaining EXPLORs according to the present invention.
FIG. 9 is an immunoblot analysis image showing the result of measurement of a change in the content of a target protein (mCherry protein) captured inside the exosome according to the intensity of blue light.
FIG. 10 is an electron micrograph showing the result of treating target cells (HT1080 cells) treated with exosome containing a target protein (mCherry) and confirming the introduction of a target protein into the target cells. And the right side represents the target cell treated with exosome.
FIG. 11 is a graph showing the fluorescence image (a) showing the result of examining whether the target protein is introduced into the target cell (HT1080 cell) after treating the exosome containing the target protein (mCherry) (B) showing the results of comparing the ratios of the apoptotic cells induced by the apoptotic cells.
FIG. 12 is a fluorescence image showing the change in intracellular location of mCherry protein according to whether blue light was irradiated in a transformant into which GIGANTEA-EGFP-CD9 gene and mCherry-FKF1LOV were introduced into HEK293T cells.
13 shows the degree of expression (a) of luciferase-mCherry fusion protein using fluorescence imaging and the number of luciferase activity and the number of molecules in the produced cells (b):
Control: HEK293T cells without any treatment;
OVER: HEK293T cells transfected with Luciferase-mCherry-CRY2 alone;
XP: HEK293T cells transfected with XPACK-Luciferase-mCherry using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the method of the present invention.
Fig. 14 is a diagram showing the measurement of the luciferase activity (a) and the number of molecules (b) in the produced exosome:
NEG: Exosomes produced in HEK293T cells that were not treated with anything;
OVER: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2;
XP: Exosomes produced in HEK293T cells using XPACK (Luciferase-mCherry XPACK) using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;
ON: Exosomes produced by culturing in 200 μW of blue light for 72 hours,
OFF: Exosome produced by culturing for 72 hours in the absence of light.
FIG. 15 is a diagram showing the loading efficiency of a target protein in the produced exosome. FIG.
FIG. 16 shows the efficiency of target protein transfer to target cells (HeLa) using exosomes:
Control: Exosomes produced in HEK293T cells without any treatment;
OVER: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2;
XP: Exosomes produced in HEK293T cells using XPACK (Luciferase-mCherry XPACK) using XPACK (Systems Biosciences), a commercial vector designed for exosome loading technology;
EXPLOR: Exosomes produced in HEK293T cells transfected with Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;
ON: Exosomes produced by culturing in 200 μW of blue light for 72 hours,
OFF: Exosome produced by culturing for 72 hours in the absence of light.
FIG. 17 shows that Luciferase-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed in the same position in HEK293T cells.
FIG. 18 shows that super-repressor-IκB-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed in the same position in HEK293T cells.
FIG. 19 is a graph showing inhibition of TNF-α-activated NF-kB migration to the nucleus and inhibition of DNA binding of NF-kB when the super-repressor-IκB: EXPLOR according to the present invention was pretreated with HeLa cells (Scale bars, 20 μm):
mCherry: EXPLOR: exosome without super-repressor-IκB; And
srlkB: EXPLOR: super-repressor-IκB: EXPLOR with super-repressor-IκB.
FIG. 20 shows the results of analysis of disease progression after administration of super repressor IκB: EXPLOR in a mouse model of rheumatoid arthritis (↓ at the time of EXPLOR administration).

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for efficiently producing a large amount of exosome containing a protein inhibiting NF-κB.

The present inventors focused on exosome-specific markers (CD9, CD63, CD81 or CD82) while carrying out various studies in order to develop a method for more effectively producing exosomes containing the target protein therein. Since the exosome-specific marker has a common property that it is a membrane protein of 4-fold penetration type belonging to the tetraspanin family, when the desired protein is bound to the membrane protein of the exosome, it can be relatively easily contained in the exosome Respectively.

The exosome-specific marker that is specifically present in the exosome membrane, which is specifically present in the exosome membrane, and the exosome-specific marker and the fusion protein of the target protein are expressed in a cell that mass-produces the exosome, a large amount of the exosome containing the target protein can be produced .

Specifically, a method for producing an exosome comprising a protein that inhibits NF-κB, which is a target protein of the present invention, is a method for producing a fusion protein comprising exosome-specific markers and a protein binding to a protein that inhibits NF-κB And introducing a polynucleotide to express it.

The resulting exosome is fused with an exosome-specific marker in which a protein that inhibits NF-κB is embedded in the exosomal membrane.

The target protein is bound to the membrane protein of the exosome and is not separated even after reaching the target cell. In order to solve these problems, various studies have been conducted. As a result, it has been found that, by using a photo-specific binding protein, a technology for temporarily producing an exosome containing the target protein by binding the target protein with the marker protein Respectively. For example, CIBN and CRY2, which are light specific binding proteins, can be used. Specifically, CIBN is expressed in a form fused with CD9, which is one of the marker proteins, and CRY2 is fused with a target protein A gene encoding these fusion proteins was introduced into the exosome-producing cells. The CIBN-CD9 fusion protein expressed in the exosome-producing cell is included in the exosome due to CD9. When the cell is irradiated with blue LED light, the target protein-CRY2 fusion protein expressed in the exosome- Is bound to the CIBN domain fused to CD9, a reversibly derived fusion protein of the target protein -CRY2-CIBN-CD9 form is formed, and such fusion protein is contained in the exosome due to CD9. When the blue light LED illumination is no longer irradiated after the exosome containing the target protein is produced, the CIBN-CRY2 binding is released and the target protein is not bound to the cell membrane of the exosome, , So that an exosome containing the target protein can be produced as a result (FIGS. 5 to 10).

The exosome thus produced may exhibit a completely different effect from the exosome including the conventional target substance. Since the conventional exosome has been expressed in the form of being fused to the exosome-specific marker in order to trap the target protein inside the exosome, the target protein is trapped in the exosome, but is still attached to the exosome membrane The target protein can be transferred to the target cell only when the exosome is fused to the cell membrane of the target cell. Thus, even when the target protein is fused to the target cell, the target protein can be bound to the exosome fused to the exosome membrane , There is a limitation that the target protein has a very low probability of exhibiting its effect in the target cell. However, since the exosome provided in the present invention is trapped in the exosome without the target protein bound to the membrane, when the exosome is endocytosed by the target cell and enters the cytoplasm, the exosome is attached to the exosome membrane Therefore, when the exosome is degraded, the target protein can be transferred to the cytoplasm of the target cell, and the target protein can freely move within the cytoplasm of the target cell, so that the target protein has an advantage that it can sufficiently exhibit the physiological activity in the target cytoplasm ( 11).

In addition, since the binding level of the target protein to be bound to the marker protein is changed according to the intensity of the light irradiated upon light irradiation, the content of the target protein captured in the exosome can be controlled when the light intensity is controlled .

Therefore, a method for producing an exosome containing a target protein using a photo-specific binding protein has not been known at all and has been developed for the first time by the present inventors.

Accordingly, the present invention provides a method for producing an exosome comprising a protein that inhibits NF-κB comprising the following steps:

(a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide encoding the first specific binding protein Introducing a polynucleotide encoding a fusion protein (second fusion protein) in which a second photo-specific binding protein capable of binding to NF-κB is bound to a protein that inhibits NF-κB;

(b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And

(c) after the exosome is produced in the exosome-producing cell, stopping the irradiation of the light.

The term "exosome " of the present invention means a small vesicle of a plasma membrane structure originating from a specific compartment within a cell called multivesicular bodies (MVBs) and released or secreted out of the cell.

In the present invention, the exosome may serve as a carrier for transporting a target protein to a target cell or tissue of a target protein by including a target protein therein. The target protein carried by the exosome may be a target cell Or may be used to treat certain diseases or to diagnose certain diseases by acting on the tissues.

The term "exosome-producing cell" of the present invention means a cell capable of producing the exosome.

In the present invention, the exosome-producing cell may be, but not limited to, a B-lymphocyte, a T-lymphocyte, a dendritic cell, a megakaryocyte, a macrophage, a stem cell, . For example, in the example of the present invention, HEK293T cells, which are a kind of immortalized cell line, were used as the exosome-producing cells.

The term "exosome-specific marker " of the present invention means a protein abundantly present in the membrane of the exosome.

In the present invention, the exosome-specific marker may be, for example, CD9, CD63, CD81, CD82, and the like. For example, in the examples of the present invention, CD9 was used as an exosome-specific marker. CD9, CD63, CD81, and CD82 are 4-fold penetration type membrane proteins. When the desired protein is bound to the exosome membrane protein, the desired protein is easily present in the exosome.

The term "photo-specific binding protein" of the present invention is also referred to as a light-oil-type heterodimer forming protein or a photoinduced homodimer forming protein. When light of a specific wavelength is irradiated, it may bind to a different protein to form a heterodimer Refers to a protein capable of binding to a protein or another protein of the same kind to form a homodimer.

In the present invention, the photo-specific binding protein may be, for example, a light oil-releasing duplex forming protein, but not limited thereto. As another example, a cryptochrome-interacting basic-helix-loop-helix protein, CIBN, phytochrome B, phytochrome interacting factor, FKF1, GIGANTEA, CRY (chryptochrome), PHR (phytolyase homolgous) region), and the like.

In particular, when forming a heterodimer, two photo-specific binding proteins (a first photo-specific binding protein and a second photo-specific binding protein) may be used, wherein the first photo-specific binding protein is CIB or CIBN , The second photo-specific binding protein may be CRY or PHR, and when the first photo-specific binding protein is PhyB, the second photo-specific binding protein may be PIF, When the first photo-specific binding protein is GIGANTEA, the second photo-specific binding protein may be FKF1.

For example, in an embodiment of the present invention, CIBN is used as the first photo-specific binding protein, CRY2 is used as the second photo-specific binding protein, and the wavelength of the used light is set to 460 to 490 nm And the intensity of the light was set to 20 to 50 μW.

Meanwhile, in order to confirm the expression of the first fusion protein in which the exosome-specific marker and the first photo-specific binding protein are bound and the position in the cell, marker proteins may be fused together. For example, in an embodiment of the present invention, expression of the first fusion protein in which CIBN and CD9, or GIGANTEA and CD9 are combined, in a form in which the fluorescent protein EGFP is inserted, And to confirm the intracellular location.

The term "target protein" of the present invention means a protein expressed in a form fused with the second photo-specific binding protein to be contained in the exosome.

In the present invention, the target protein is a protein that inhibits NF-κB. The protein that inhibits NF-κB is a super-repressor-IκB protein and binds to NF-κB to be inactivated in the cytoplasm.

The super-repressor-IκB protein is a S32A, S36A mutant form of IκB that is not phosphorylated by IκB kinase (IKK) and is not degraded by proteasome, resulting in the ability to continuously inhibit NF-κB Function.

The protein that inhibits NF-κB, which is a target protein of the present invention, is preferably one or more selected from the group consisting of proteins represented by amino acid sequences of any one of SEQ ID NOs: 1 to 5, but is not limited thereto, and IκB-α , IκB-β, IκB-ε, BCL-3, or mutants thereof, but it is possible that the protein can inhibit NF-κB.

The term "cultivation" of the present invention means a method of growing a cell or a microorganism under an appropriately artificially controlled environmental condition.

In the present invention, the transformant is cultivated for 1 to 3 days, then replaced with a medium not containing fetal bovine serum, and further cultured for 2 to 5 days.

In the present invention, the method for culturing the transformant may be carried out by a method well known in the art.

The medium means a known medium used for animal cell culture and may be selected from a group such as commercially available serum free media, protein free media and chemically defined media .

Preferably, the serum-free medium is SFM4CHO (HyClone), EX-Cell (JHR Bioscience) or the like, and the insulin-like growth factor I -I), ethanolamine, ferric chloride, and phosphatidyl choline may be added, but the present invention is not limited thereto.

The animal cell culture medium in which the protein derived from animal, in particular, the protein having a molecular weight of 10 kDa or more is removed in the serum-free medium from which the bovine serum component has been removed, is selected as Prostate (Lonza) and PF-CHO (HyClone) But is not limited thereto.

The chemically defined medium is an animal cell culture medium having no known animal-derived components and all constituent components of which are known chemical structures. These media include CDM4CHO (HyClone), PowerCHO2CD (Lonza), and CD-optiCHO ) May be selected, but the present invention is not limited thereto.

The term "first fusion protein" of the present invention means a fusion protein in which the exosome-specific marker and the first photo-specific binding protein are bound.

In the present invention, the sequence of the exosome-specific marker and the first photo-specific binding protein contained in the first fusion protein is such that the first fusion protein binds to the first light in the exosome- As long as it can be expressed so that the specific binding protein can be expressed so as to be positioned so that the N-terminal of the first photo-specific binding protein is bound to the C-terminus of the exosome-specific marker .

The exosome-specific marker constituting the first fusion protein and the first photo-specific binding protein may be directly linked to each other, or may be linked through a linker. The linker is not particularly limited as long as the first fusion protein can be expressed so that the first photo-specific binding protein is located in the exosome-producing cell in the inside of the exosome-producing cell, but a peptide linker composed of an amino acid can be used And more preferably a flexible peptide linker can be used. The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.

The term "second fusion protein" of the present invention means a fusion protein in which the second photo-specific binding protein and the target protein are combined.

In the present invention, the order of arrangement of the second photo-specific binding protein and the target protein contained in the second fusion protein is such that the second fusion protein is the first photo-specific of the first fusion protein in the exosome- As long as it can bind to the binding protein site and be located within the exosome, it is not particularly limited. For example, the N-terminal of the target protein is bound to the C-terminus of the second photo- specific binding protein .

In addition, the second photo-specific binding protein and the target protein constructing the second fusion protein may be directly coupled to each other, or may be connected to each other through a linker. The linker is not particularly limited as long as the second fusion protein can be located in the exosome by binding to the first photo-specific binding protein region of the first fusion protein in the exosome-producing cell, Linkers can be used, and more preferably, flexible peptide linkers can be used. The peptide linker may be operably linked to an expression vector by linking the nucleic acid encoding the linker in-frame between the nucleic acids encoding each domain.

In addition, each of the fusion proteins may include a polypeptide having a sequence that differs from a wild-type amino acid sequence of each domain contained therein by one or more amino acid residues. Amino acid exchange in proteins and polypeptides that do not globally alter the activity of the molecule is known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity due to mutation or modification of the amino acid sequence, such as heat, pH and the like.

Finally, the polypeptide of each domain constituting the fusion protein or the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or may be prepared by amplifying a gene encoding the domain by PCR (polymerase chain reaction) Followed by expression and cloning into an expression vector.

On the other hand, each fusion protein can be expressed in the exosome-producing cell by introducing a polynucleotide encoding the fusion protein into the exosome-producing cell. As the method for introducing the polynucleotide into the exosome-producing cell, Method can be used. For example, a method of introducing using an expression vector can be used.

The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene product containing an essential regulatory element operatively linked to the expression of the gene insert. The expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc. The initiation codon and termination codon are generally regarded as part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid sequence encoding a desired protein or RNA is functionally linked to a nucleic acid expression control sequence so as to perform a general function . For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with an expression vector can be produced using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage can be performed using enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide to facilitate the separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG start codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic sources. Expression efficiency can be increased by the introduction of suitable transcription or translation enhancers.

According to a preferred embodiment of the present invention, the expression vector can be expressed by binding a tag to the target protein to confirm whether the target protein is inserted into the exosome. Since the tag is for confirming the presence or absence of the target protein, the target protein can be bound to the opposite site to which the second photo-specific binding protein is bound. For example, fluorescence of a red fluorescent protein, The protein can be used as a tag to bind to the C-terminal region of the target protein.

It is possible to confirm whether or not the exosome contains the target protein by expressing the designed protein in the exosome-producing cell and confirming whether the fluorescent protein tag is detected in the exosome after the exosome is produced .

The term "light" of the present invention means light irradiated to temporarily bind the first light-specific binding protein and the second light-specific binding protein expressed in the exosome-producing cell.

As described above, in the exosome-producing cell, the first photo-specific binding protein is expressed in the form of the first fusion protein together with the exosome-specific marker, and the second photo-specific binding protein is expressed in the form of the second fusion protein Specific binding protein is irradiated with light necessary for binding of the first specific binding protein and the second specific binding protein to the exosome-producing cell, the first specific binding protein and the second specific Binding protein is bound and consequently a fusion protein complex having the shape of the exosome-specific marker-first light-specific binding protein-second light-specific binding protein-target protein is temporarily formed. The exosome-producing cell , The target protein may be linked to the exosome due to the exosome-specific marker. In this case, the target protein is present inside the exosome, and when irradiation of light is stopped after the exosome is produced, the first photo-specific binding protein and the second photo-specific binding protein are separated, The target protein present in the inside is released to the outside in the form contained in the exosome upon the release of the exosome. It is also preferable that the light is intermittently irradiated rather than continuously irradiated so that the target protein can be more efficiently introduced into the exosome. That is, when light is intermittently irradiated, the binding and separation of the first photo-specific binding protein and the second photo-specific binding protein are repeated, so that the probability that the target protein is introduced into the exosome can be improved .

Meanwhile, since the wavelength of the light that induces the binding of the first photo-specific binding protein and the second photo-specific binding protein depends on the kind of the first photo-specific binding protein and the second photo-specific binding protein, The wavelength of the light can be selected. That is, when CRY2 and CIBN are combined, light having a wavelength of 460 to 490 nm is irradiated, and when the light is not irradiated for 10 minutes or more, CRY2 and CIBN are dissociated from each other; When PhyB and PIF are combined, light having a wavelength of 650 nm is irradiated for 10 minutes, and light having a wavelength of 750 nm is irradiated for 5 minutes, PhyB and PIF dissociate from each other; When combining FKF1 and GIGANTEA, light with a wavelength of 460 nm can be irradiated for 30 minutes. In the embodiment of the present invention, light having a wavelength of 460 to 490 nm was irradiated to induce the binding of CIBN and CRY2.

According to the embodiment of the present invention, the expression of the fusion protein of CRY2 and mCherry and the fusion protein of CIB and CD9 was expressed in HEK293T cell, an immortal cell line producing a large amount of exosome. As a result, the distribution of mCherry protein uniformly distributed in cytoplasm When irradiated with blue light, it was observed that it is in a membrane such as a cell membrane and an endosome-like structure (Fig. 7). Similar results were also observed when FKF1 and mCherry fusion proteins and GIGANTEA and CD9 fusion proteins were expressed in HEK293T cells (Fig. 12). Furthermore, when the fusion proteins of CRY2 and mCherry and the fusion proteins of CIBN and CD9 were expressed in HEK293T and the intensity of blue light was controlled, it was found that mCherry protein captured in the exosomes (FIG. 9). As a result of treatment of HT1080 cells, which are isolated from exocytes produced from the cells, at a concentration of about 250 μg / ml, they did not show any cytotoxicity against HT1080 cells, MCherry protein was transferred to the cytoplasm of HT1080 cells (Fig. 10).

In order to compare the introduction efficiency of the target protein in the exosome of the present invention and the exosome transfer efficiency to the target cell with the conventional method, XPACK vector was used as the conventional method, and the CRY2 and mCherry proteins of the present invention Type fusion protein with CIBN and CD9 protein was introduced into HEK293T, and the amount of target protein produced in the exosome was compared. As a result, when the method of the present invention was used, (Fig. 15). In addition, when the exosome isolated from the exosome-producing cell is treated with target cells (HeLa) and the degree of expression of the target protein is compared, when the exosome isolated by the method of the present invention is used, And the highest expression was confirmed (Fig. 16).

The present invention provides, in another aspect, (a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first photo-specific binding protein and an exosome-specific marker are bound; And (b) a polynucleotide encoding a multicloning site into which a polynucleotide encoding a target protein can be introduced and a second photo-specific binding protein capable of binding to the first photo-specific binding protein, And a second expression vector comprising the exogenous expression vector.

In the vector for producing an exosome provided by the present invention, the exosome-specific marker, the first photo-specific binding protein, the exosome-producing cell and the second photo-specific binding protein are the same as described above.

The term "transforming cell for producing exosome" of the present invention refers to a cell that encodes a fusion protein (first fusion protein) in which exosome-specific markers and a first photo-specific binding protein are bound to the exosome- Refers to an exosome-producing cell into which a nucleotide has been introduced to express the first fusion protein.

In the present invention, the second expression vector may include a polynucleotide encoding the second photo-specific binding protein, and may be constructed to include a polyclonal region adjacent to the polynucleotide. The second expression vector When a polynucleotide encoding a target protein is introduced into the polyclonal region, the second light specific binding protein and the target protein can be expressed as a fusion form (second fusion protein).

The vector for producing exosome provided by the present invention can be used not only for transfecting cells and expression vectors for production of exosome but also for introducing the expression vector, culturing transgenic cells for production of exosome, producing transgenic cells for producing exosome One or more other component compositions, solutions or devices suitable for isolating and purifying exosomes may also be included. For example, it may further comprise an appropriate buffer necessary for introduction of the expression vector, a medium and a container necessary for culturing the transformed cell for producing the exosome, and the like.

In another aspect, the present invention provides an exosome produced by the above method and comprising a protein that inhibits NF-kB.

The exosome produced by the above-mentioned method contains a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the plasma membrane constituting the exosome, (Second fusion protein) in which a second photo-specific binding protein capable of binding to the first photo-specific binding protein and a protein inhibiting NF-κB are combined, the exosome Upon treatment with cells in the target tissue, the second fusion protein contained in the exosome can be transferred to the cytoplasm in the target tissue through fusion of the plasma membrane.

The present invention also provides a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases which comprises an exosome carrying an NF-κB inhibiting protein prepared by the above method as an active ingredient.

The inflammatory disease is selected from the group consisting of allergies, dermatitis, atopic dermatitis, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, , Ulcerative colitis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, acute and chronic inflammatory diseases, sepsis, ulcerative colitis, and Crohns disease, but is not limited thereto.

In a specific example of the present invention, the inventors of the present invention conducted a study to confirm the inhibitory effect of TNF-α mediated inflammation using super-repressor-IκB-loaded exoposome (super-repressor-IκB: EXPLOR) The super-repressor-IκB: EXPLOR was pretreated with HeLa cells, confirming that the migration of activated NF-κB to the nucleus by TNF-α was inhibited (see FIG. 19A). In addition, it was confirmed that DNA binding of NF-κB activated by TNF-α was suppressed by pretreating super-repressor-IκB: EXPLOR (see FIG. 19B).

In order to confirm the inhibitory effect of the super-repressor-IκB: EXPLOR prepared in the above-described manner on the collagen-induced arthritic rat model, the present inventors used collagen-induced arthritis animal model of super repressor IκB: EXPLOR And intravenous injection of orbital and arthritis-induced rats showed decreased arthritic symptoms (see FIG. 20).

Therefore, the super-repressor-I? B-loaded exoposum of the present invention (super-repressor-IκB: EXPLOR) can be used as a pharmaceutical composition for the prevention and treatment of inflammatory diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Exosome  Produce

<1-1> Confirmation of binding of CIBN and CRY2 for the production of exosome

The pcDNA3.1 (+) vector containing the CIBN-EGFP-CD9 gene and the pcDNA3.1 (+) vector containing the mCherry-CRY2 gene were introduced into the exosome-producing HEK293T cells under light-free conditions, , Then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the cultivation, blue light having a wavelength of 460 to 490 nm was irradiated, and the positions of red fluorescence appearing in the mCherry before and after irradiation of the blue light were confirmed through a confocal microscope (Fig. 7).

FIG. 7 is a fluorescence photograph showing the change in the intracellular location of mCherry protein in a transformant into which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells according to irradiation of blue light. As shown in FIG. 7, mCherry protein is uniformly distributed in the cytoplasm before blue light that induces the binding of CIBN and CRY2, which is a photo-specific binding protein, is observed. However, after the blue light is irradiated, Of the total population. This clustering of mCherry protein was analyzed to be caused by the combination of CIBN and CRY2, a light specific binding protein.

<1-2> Confirmation of binding of GIGANTEA and FKF1 for the production of exosome

Using pcDNA3.1 (+) vector containing GIGANTEA-EGFP-CD9 gene and pcDNA3.1 (+) vector containing mCherry-FKF1LOV gene and using the same method as in Example <1-1> Exosomes in the cells were confirmed. (In the above FKF1LOV, LOV is an abbreviation of light-oxygen-voltage domain. It indicates a domain in which FKF1 protein actually binds to another protein by light. Thus, FKF1 and FKF1LOV are used in the same meaning.)

As shown in FIG. 12, the mCherry protein is uniformly distributed in the cytoplasm before the blue light that induces the binding of GIGANTEA and FKF1, which is a photo-specific binding protein, is irradiated. However, The mCherry protein was dense in the membrane (Fig. 12). Thus, similar to the results of <1-1>, it was confirmed that the dense mCherry protein can be induced by the binding of GIGANTEA and FKF1 of the photo-specific binding protein.

Exosome  Production and Exosome  Effect of light intensity on production

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene, respectively, was irradiated with light of a wavelength of 460 nm at an intensity of 0, 5, 20, 50 or 200 μW, , HEK293T cells, cultured for 24 hours, then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the culture, the culture broth was separated and centrifuged (3000 × g, 15 minutes) to obtain a supernatant from which cellular debris was removed. The obtained supernatant was mixed with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, California, USA) of 5 times the volume of the supernatant and centrifuged (1500 x g, 30 minutes) Cotton was obtained, and the resulting exosome was suspended in PBS to give an exosome suspension. The exosome suspension was filtered through a 0.2 탆 filter using a syringe equipped with a 27-G needle to obtain a single-sized exosome (Fig. 8). Then, Exosome Lysate was prepared using Lysis buffer, and the amount of mCherry protein contained in exosome was compared through immunoblot analysis (FIG. 9).

FIG. 9 is an immunoblot analysis image showing the result of measurement of a change in the content of a target protein (mCherry protein) captured inside the exosome according to the intensity of blue light. As shown in FIG. 9, when the blue light was irradiated at an intensity of 20 to 50 μW, it was confirmed that the content of the target protein (mCherry protein) captured in the exosome exhibited the maximum value. From the above results, it was found that the content of the target protein captured in the exosome can be controlled by controlling the intensity of the light irradiated upon binding of the photo-specific binding protein.

Exosome  Treatment effect

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, under the LEDs that emit light with a wavelength of 460 nm at an intensity of 50 μW Exosome was extracted by the method of Example 2. Then, the extracted exosome was treated with HT1080 cells at a concentration of 250 μg / ml for 24 hours. Then, the HT1080 cells were fixed with 0.1 M phosphate buffer (pH 7.4) containing 4% PFA and 0.01% GA and fixed on 10% gelatin gel. The cells adhered onto the gelatin gel were cooled with liquid nitrogen for one day and a slice cut to a thickness of 45 nm was obtained at -120 캜 using a cryoultramicrotome. Next, the flakes were immunostained with anti-mCherry antibody and protein A-gold, and mCherry protein was observed through Tecnai G2 Spirit Twin TEM (FIG. 10).

FIG. 10 is an electron micrograph showing the result of treating target cells (HT1080 cells) treated with an exosome containing a target protein (mCherry) and confirming the introduction of a target protein into the target cell. And the right side represents the target cell treated with exosome. As shown in FIG. 10, when the exosome of the present invention was treated on the target cell, it was confirmed that the target protein was transferred into the target cell.

Containing the desired protein Exosome  analysis

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene was introduced into HEK293T cells, which are exosome-producing cells, under the LEDs that emit light with a wavelength of 460 nm at an intensity of 50 μW Exosome was extracted by the method of Example 2. Then, the extracted exosome was treated with HT1080 cells at a concentration of 250 μg / ml for 24 hours. Then, the red fluorescence of mCherry protein was confirmed by fluorescence microscopy, and the ratio of dead cells of cells treated with exosome and untreated cells was compared through LDH cell death assay (FIG. 11).

FIG. 11 is a graph showing fluorescence (a) showing the result of examining whether the target protein is introduced into the target cell (HT1080 cell) after treating the exosome containing the target protein (mCherry) to the target cell (B) showing the results obtained by comparing the ratios of the apoptotic cells induced by the apoptotic cells. As shown in Fig. 11, it was confirmed that apoptosis did not induce apoptosis.

Exosome  Produced and produced Exosome  Comparison of introduction efficiency of target protein

<5-1> Confirmation of exosome production efficiency

In order to confirm the expression of the target protein in the exosome-producing cells in order to compare the exosome production of the present invention and the degree of introduction of the target protein in the produced exosome, the luciferase activity measurement experiment was performed .

In the conventional method, XPACK-Luciferase-mCherry was introduced into HEK293T cells (XP) using XPACK (Systems Biosciences), which is a commercial vector designed for exosome mounting technology, and Luciferase-mCherry-CRY2 And CIBN-EGFP-CD9 were introduced into HEK293T cells (EXPLOR), and then the intracellular luciferase activity was measured to compare the efficiency of the two methods. The luciferase activity was measured according to the manufacturer's instructions (Luciferase Assay Reagent, Promega), and the standard curve of the result was plotted, and then the number of exocytes in the cell was quantitatively calculated.

As shown in FIG. 14, it was confirmed that the method using the photo-specific binding proteins CIBN and CRY2 of the present invention was significantly higher in the introduction efficiency into exosomes than the conventional method XP (FIG. 14).

<5-2> Expression of the target protein in the produced exosome

The cells of the above Example <5-1> were cultured for 72 hours, and the exosomes were separated (Exoquick-TC, Systems biosciences) As a result of indirectly comparing the amount of the target protein with the activity of luciferase activity, as shown in FIG. 15, the method of the present invention can produce an exosome containing a remarkably large amount of the target protein (Fig. 15).

<5-3> Comparison of introduction efficiency of target protein

Using the measured luciferase activity values in the above Examples <5-1> and <5-2>, the introduction efficiency (E) of the target protein was calculated by the following equation (1).

[Equation 1]

As shown in FIG. 15, it was confirmed that the exosomes produced using the CRY2 and CIBN binding of the present invention were 4 to 120 times more efficient than the other comparative groups (FIG. 15).

Target cell Exosome  Comparison of transfer efficiency

In order to compare the efficiency of treatment of the exosome into which the target protein was introduced to the target cells, 5 × 10 ⁹ exosomes were treated with HeLa cells for 24 hours and the fluorescence intensity expressed in the cells was measured. As a result, As shown, it was confirmed that the fluorescence intensity of EXPLOR of the present invention was remarkably high (FIG. 16).

Therefore, it can be seen that the method of using the exosome of the present invention can more effectively deliver the target protein to the target cell than the conventional method.

< Experimental Example  1> super-repressor-IκB end  Mounted Exosome (super-repressor-IKB : EXPLOR )

<1-1> Exosome  The internal super-repressor- IκB  Protein identification

In order to confirm that the super-repressor-IκB protein described in SEQ ID NO: 5 is loaded in the exosome, the present inventors used CIBN-EGFP-CD9 and super-repressor-IκB-mCherry- And CRY2.

Specifically, the pcDNA3.1 (+) vector containing the CIBN-EGFP-CD9 gene and the pcDNA3.1 (+) vector containing the super-repressor-IκB-mCherry-CRY2 gene under the light- , HEK293T cells, cultured for 24 hours, then replaced with medium without fetal bovine serum, and further cultured for 48 hours. After completion of the cultivation, blue light having a wavelength of 488 nm was irradiated, and the positions of red fluorescence appearing in the mCherry before and after irradiation of the blue light were confirmed through a confocal microscope. The experiment was repeated 5 or more times.

As a result, it was confirmed that super-repressor-IκB-mCherry-CRY2 (red) and CIBN-EGFP-CD9 (green) were co-localized by yellow light stimulation (yellow) 18). This confirms that the super-repressor-IκB protein was loaded inside the exosome.

<2-2> super-repressor- IκB  Protein-loaded Exosome (super-repressor- IκB: EXPLOR ) Production

The present inventors conducted the following experiment to obtain an exosome carrying the super-repressor-I [kappa] B protein.

Specifically, each expression vector containing the CIBN-EGFP-CD9 gene and the super-repressor-IκB-mCherry-CRY2 gene was introduced into the exosome-producing HEK293T cells and cultured for 24 hours. Then, fetal calf serum And then cultured for 48 hours at an intensity of 50 μW under an LED illuminating light of 488 nm wavelength. After the incubation was completed, the culture broth was separated and centrifuged (2000 xg, 15 minutes) to obtain a supernatant from which cellular debris was removed. To the supernatant obtained above, 5 times volume of the supernatant was mixed with ExoQuick-TC Exosome Precipitation Solution (System Biosciences, Mountain View, California, USA) at 4 ° C for 18 hours, centrifuged (1500 × g, 30 minutes) to obtain a precipitated exosome. The resulting exosome was suspended in PBS to obtain an exosome suspension (FIG. 8).

In addition, for the mass production of exosomes, HEK293T cells, which are exosome-producing cells that stably express the respective expression vectors containing the CIBN-EGFP-CD9 gene and the super-repressor-IκB-mCherry-CRY2 gene, And cultured in a medium containing no serum for 48 to 72 hours under an LED emitting light of 488 nm at an intensity of 50 μW. After the incubation was completed, the culture broth was separated and centrifuged (2000 xg, 15 minutes) to obtain a supernatant from which cellular debris was removed. It was filtered with 0.2 ul PES membrane (Corning) to remove particles above 200 nm from the supernatant. Tangential Flow Filtration (TFF) filtration was used to remove particles below 20 nm from the same filtrate and to concentrate and purify the exosome from the filtrate. The membrane of TFF was a Vivaflow 50-100 KDa PES membrane (Sartorius). The filtrate was repeatedly rotated at a TFF air pressure of 1.5 to 2 to concentrate and purify the exosome. After that, the exocomma concentrate was transferred to an Amicon Ultra-0.5 (100 kDa) (Millipore) filter and centrifuged at 10,000 to 14,000 g for 5 minutes to remove the aqueous solution. And centrifuged for 5 minutes to obtain the final exosome.

< Experimental Example  2> super-repressor-IκB end  Mounted Exosome (super-repressor-IKB : EXPLOR ) Inhibited TNF-α mediated NF-κB activity

The present inventors carried out the following experiments to confirm the TNF-α mediated inflammation-suppressing effect using the super-repressor-IκB: EXPLOR prepared in the above <Experimental Example 1>.

Specifically, HeLa cells were cultured in medium containing 100 mg / mL of mCherry: EXPLORs or super-repressor-IκB-mCherry: EXPLORs for 3 hours. And then treated with TNF-α (10 ng / mL) for an additional 30 min. After fixation with 4% paraformaldehyde, NF-κB p65 was stained with Alexa Fluor 488 conjugated antibody and observed by confocal microscopy. To determine the binding activity of p65 / c-Rel (NF-kB), nuclear extracts were prepared according to the manual of TransAM NF-kB and AP-1 assay kit (ActiveMotif, Carlsbad, CA, USA). Results Data were expressed as mean ± SEM (n = 3), and significant group effects (**, p <0.01) were assessed by ANOVA using Tukey's post hoc test.

As a result, as shown in FIG. 19, super-repressor-IκB: EXPLOR was pretreated with HeLa cells, and it was confirmed that the migration of activated NF-κB to the nucleus by TNF-α was inhibited (FIG. In addition, it was confirmed that DNA binding of NF-κB activated by TNF-α was inhibited by pretreating super-repressor-IκB: EXPLOR (FIG. 19B).

< Experimental Example  3> Collagen induction-arthritis In animal models super-repressor- IκB  Mounted Exosome (super-repressor-IκB: EXPLOR)

The present inventors conducted the following experiments to confirm the inhibitory effect of the super-repressor-IκB: EXPLOR prepared in <Experimental Example 1> on collagen-induced arthritic rat model.

Specifically, a collagen-induced arthritis rat model most commonly used in rheumatoid arthritis studies was constructed by injecting bovine collagen type II and adjuvant into tail subcutaneously in DBA / 1 mice. Two mice with arthritis were injected with super repressor IκB: EXPLOR intravenously four times at intervals of two days each. The progression of rheumatoid arthritis symptoms was judged based on symptom scores as shown in Table 1 according to severity of symptoms. The Mean Clinical Score is the mean value of each symptom score of the rats given the above criteria.

As a result, as shown in FIG. 20, when the super repressor IκB: EXPLOR was intravenously injected three times, it was confirmed that arthritis symptoms were reduced in the mice induced with arthritis.

Symptom score Pathology type 0 No erythema and swelling One Erythema and mild swelling of ankle or ankle joints were identified. 2 Erythema and slight swelling from ankles to ankle bones expanded 3 Erythema and normal swelling from ankle to footbone expanded 4 Ankle, foot and fingers as well as erythema and severe swelling,

<110> Korea Advanced Institute of Science and Technology          Cellex Life Sciences Inc. <120> Process for preparing exosome loading super-repressor-IkB          protein, and pharmaceutical composition for use in preventing or          inflammatory diseases containing the same as an active          ingredient <130> 2016P-05-042 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 317 <212> PRT <213> Homo sapiens <400> 1 Met Phe Gln Ala Ala Glu Arg Pro Gln Glu Trp Ala Met Glu Gly Pro   1 5 10 15 Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Hisp Ser              20 25 30 Gly Leu Asp Ser Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu          35 40 45 Leu Gln Glu Ile Arg Leu Glu Pro Gln Glu Val Pro Arg Gly Ser Glu      50 55 60 Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu  65 70 75 80 Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Ile Arg Gln                  85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln             100 105 110 Thr 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295 300 Tyr Asp Asp Cys Val Phe Gly Gly Gln Arg Leu Thr Leu 305 310 315 <210> 2 <211> 356 <212> PRT <213> Homo sapiens <400> 2 Met Ala Gly Val Ala Cys Leu Gly Lys Ala Ala Asp Ala Asp Glu Trp   1 5 10 15 Cys Asp Ser Gly Leu Gly Ser Leu Gly Pro Asp Ala Ala Ala Pro Gly              20 25 30 Gly Pro Gly Leu Gly Ala Glu Leu Gly Pro Gly Leu Ser Trp Ala Pro          35 40 45 Leu Val Phe Gly Tyr Val Thr Glu Asp Gly Asp Thr Ala Leu His Leu      50 55 60 Ala Val Ile His Gln His Glu Pro Phe Leu Asp Phe Leu Leu Gly Phe  65 70 75 80 Ser Ala Gly Thr Glu Tyr Met Asp Leu Gln Asn Asp Leu Gly Gln Thr                  85 90 95 Ala Leu His Leu Ala Ala Ile Leu Gly Glu Thr Ser Thr Val Glu Lys             100 105 110 Leu Tyr Ala Gly Ala Gly Leu Cys Val Ala Glu Arg Arg Gly His         115 120 125 Thr Ala Leu His Leu Ala Cys Arg Val Gly Ala His Ala Cys Ala Arg     130 135 140 Ala Leu Leu Gln Pro Arg Pro Arg Arg Pro Glu Ala Pro Asp Thr 145 150 155 160 Tyr Leu Ala Gln Gly Pro Asp Arg Thr Pro Asp Thr Asn His Thr Pro                 165 170 175 Val Ala Leu Tyr Pro Asp Ser Asp Leu Glu Lys Glu Glu Glu Glu Ser             180 185 190 Glu Glu Asp Trp Lys Leu Gln Leu Glu Ala Glu Asn Tyr Glu Gly His         195 200 205 Thr Pro Leu His Val Ala Val Ile His Lys Asp Val Glu Met Val Val     210 215 220 Leu Leu Arg Asp Ala Gly Ala Asp Leu Asp Lys Pro Glu Pro Thr Cys 225 230 235 240 Gly Arg Ser Ser Leu His Leu Ala Val Glu Ala Gln Ala Ala Asp Val                 245 250 255 Leu Glu Leu Leu Leu Arg Ala Gly Ala Asn Pro Ala Ala Arg Met Tyr             260 265 270 Gly Gly Arg Thr Pro Leu Gly Ser Ala Met Leu Arg Pro Asn Pro Ile         275 280 285 Leu Ala Arg Leu Leu Arg Ala His Gly Ala Pro Glu Pro Glu Gly Glu     290 295 300 Asp Glu Lys Ser Gly Pro Cys Ser Ser Ser Ser Asp Ser Asp Ser Gly 305 310 315 320 Asp Glu Gly Asp Glu Tyr Asp Asp Ile Val Val His Ser Ser Arg Ser                 325 330 335 Gln Thr Arg Leu Pro Pro Thr Pro Ala Ser Lys Pro Leu Pro Asp Asp             340 345 350 Pro Arg Pro Val         355 <210> 3 <211> 500 <212> PRT <213> Homo sapiens <400> 3 Met Asn Gln Arg Arg Ser Glu Ser Arg Pro Gly Asn His Arg Leu Gln   1 5 10 15 Ala Tyr Ala Glu Pro Gly Lys Gly Asp Ser Gly Gly Ala Gly Pro Leu              20 25 30 Ser Gly Ser Ala Arg Arg Gly Arg Gly Gly Gly Gly Ala Ile Arg Val          35 40 45 Arg Arg Pro Cys Trp Ser Gly Gly Ala Gly Arg Gly Gly Gly Pro Ala      50 55 60 Trp Ala Val Arg Leu Pro Thr Val Thr Ala Gly Trp Thr Trp Pro Ala  65 70 75 80 Leu Arg Thr Leu Ser Ser Leu Arg Ala Gly Pro Ser Glu Pro His Ser                  85 90 95 Pro Gly Arg Arg Pro Pro Arg Ala Gly Arg Pro Leu Cys Gln Ala Asp             100 105 110 Pro Gln Pro Gly Lys Ala Ala Arg Arg Ser Leu Glu Pro Asp Pro Ala         115 120 125 Gln Thr Gly Pro Arg Pro Ala Arg Ala Gly Met Ser Glu Ala Arg     130 135 140 Lys Gly Pro Asp Glu Ala Glu Glu Ser Gln Tyr Asp Ser Gly Ile Glu 145 150 155 160 Ser Leu Arg Ser Leu Arg Ser Leu Pro Glu Ser Thr Ser Ala Pro Ala                 165 170 175 Ser Gly Pro Ser Asp Gly Ser Pro Gln Pro Cys Thr His Pro Pro Gly             180 185 190 Pro Val Lys Glu Pro Glu Glu Lys Glu Asp Ala Asp Gly Glu Arg Ala         195 200 205 Asp Ser Thr Tyr Gly Ser Ser Ser Leu Thr Tyr Thr Leu Ser Leu Leu     210 215 220 Gly Gly Pro Glu Ala Glu Asp Pro Ala Pro Arg Leu Pro Leu Pro His 225 230 235 240 Val Gly Ala Leu Ser Pro Gln Gln Leu Glu Ala Leu Thr Tyr Ile Ser                 245 250 255 Glu Asp Gly Asp Thr Leu Val His Leu Ala Val Ile His Glu Ala Pro             260 265 270 Ala Val Leu Leu Cys Cys Leu Ala Leu Leu Pro Gln Glu Val Leu Asp         275 280 285 Ile Gln Asn Asn Leu Tyr Gln Thr Ala Leu His Leu Ala Val His Leu     290 295 300 Asp Gln Pro Gly Ala Val Arg Ala Leu Val Leu Lys Gly Ala Ser Arg 305 310 315 320 Ala Leu Gln Asp Arg His Gly Asp Thr Ala Leu His Val Ala Cys Gln                 325 330 335 Arg Gln His Leu Ala Cys Ala Arg Cys Leu Leu Glu Gly Arg Pro Glu             340 345 350 Pro Gly Arg Gly Thr Ser His Ser Leu Asp Leu Gln Leu Gln Asn Trp         355 360 365 Gln Gly Leu Ala Cys Leu His Ile Ala Thr Leu Gln Lys Asn Gln Pro     370 375 380 Leu Met Glu Leu Leu Leu Arg Asn Gly Ala Asp Ile Asp Val Gln Glu 385 390 395 400 Gly Thr Ser Gly Lys Thr Ala Leu His Leu Ala Val Glu Thr Gln Glu                 405 410 415 Arg Gly Leu Val Gln Phe Leu Leu Gln Ala Gly Ala Gln Val Asp Ala             420 425 430 Arg Met Leu Asn Gly Cys Thr Pro Leu His Leu Ala Ala Gly Arg Gly         435 440 445 Leu Met Gly Ile Ser Ser Thr Leu Cys Lys Ala Gly Ala Asp Ser Leu     450 455 460 Leu Arg Asn Val Glu Asp Glu Thr Pro Gln Asp Leu Thr Glu Glu Ser 465 470 475 480 Leu Val Leu Leu Pro Phe Asp Asp Leu Lys Ile Ser Gly Lys Leu Leu                 485 490 495 Leu Cys Thr Asp             500 <210> 4 <211> 454 <212> PRT <213> Homo sapiens <400> 4 Met Pro Arg Cys Pro Ala Gly Ala Met Asp Glu Gly Pro Val Asp Leu   1 5 10 15 Arg Thr Arg Pro Lys Ala Gly Leu Pro Gly Ala Ala Leu Pro Leu              20 25 30 Arg Lys Arg Pro Leu Arg Ala Pro Ser Pro Glu Pro Ala Ala Pro Arg          35 40 45 Gly Ala Gly Leu Val Val Pro Leu Asp Pro Leu Arg Gly Gly Cys      50 55 60 Asp Leu Pro Ala Val Pro Gly Pro Pro His Gly Leu Ala Arg Pro Glu  65 70 75 80 Ala Leu Tyr Tyr Pro Gly Ala Leu Leu Pro Leu Tyr Pro Thr Arg Ala                  85 90 95 Met Gly Ser Pro Phe Pro Leu Val Asn Leu Pro Thr Pro Leu Tyr Pro             100 105 110 Met Met Cys Pro Met Glu His Pro Leu Ser Ala Asp Ile Ala Met Ala         115 120 125 Thr Arg Ala Asp Glu Asp Gly Asp Thr Pro Leu His Ile Ala Val Val     130 135 140 Gln Gly Asn Leu Pro Ala Val His Arg Leu Val Asn Leu Phe Gln Gln 145 150 155 160 Gly Gly Arg Glu Leu Asp Ile Tyr Asn Asn Leu Arg Gln Thr Pro Leu                 165 170 175 His Leu Ala Val Ile Thr Thr Leu Pro Ser Val Val Arg Leu Leu Val             180 185 190 Thr Ala Gly Ala Ser Pro Met Ala Leu Asp Arg His Gly Gln Thr Ala         195 200 205 Ala His Leu Ala Cys Glu His Arg Ser Pro Thr Cys Leu Arg Ala Leu     210 215 220 Leu Asp Ser Ala Ala Pro Gly Thr Leu Asp Leu Glu Ala Arg Asn Tyr 225 230 235 240 Asp Gly Leu Thr Ala Leu His Val Ala Val Asn Thr Glu Cys Gln Glu                 245 250 255 Thr Val Gln Leu Leu Leu Glu Arg Gly Ala Asp Ile Asp Ala Val Asp             260 265 270 Ile Lys Ser Gly Arg Ser Pro Leu Ile His Ala Val Glu Asn Asn Ser         275 280 285 Leu Ser Met Val Gln Leu Leu Leu Gln His Gly Ala Asn Val Asn Ala     290 295 300 Gln Met Tyr Ser Gly Ser Ser Ala Leu His Ser Ala Ser Gly Arg Gly 305 310 315 320 Leu Leu Pro Leu Val Arg Thr Leu Val Arg Ser Gly Ala Asp Ser Ser                 325 330 335 Leu Lys Asn Cys His Asn Asp Thr Pro Leu Met Val Ala Arg Ser Arg             340 345 350 Arg Val Ile Asp Ile Leu Arg Gly Lys Ala Thr Arg Pro Ala Ser Thr         355 360 365 Ser Gln Pro Asp Pro Ser Pro Asp Arg Ser Ala Asn Thr Ser Pro Glu     370 375 380 Ser Ser Ser Le Ser Ser Asn Gly Leu Ser Ser Ser Ser Ser Ser 385 390 395 400 Ser Ser Pro Ser Gln Ser Pro Pro Arg Asp Pro Pro Gly Phe Pro Met                 405 410 415 Ala Pro Pro Asn Phe Phe Leu Pro Ser Ser Pro Pro Ala Phe Leu             420 425 430 Pro Phe Ala Gly Val Leu Arg Gly Pro Gly Arg Pro Val Val Pro Ser         435 440 445 Pro Ala Pro Gly Gly Ser     450 <210> 5 <211> 317 <212> PRT <213> Artificial Sequence <220> <223> IkB alpha mutation <400> 5 Met Phe Gln Ala Ala Glu Arg Pro Gln Glu Trp Ala Met Glu Gly Pro   1 5 10 15 Arg Asp Gly Leu Lys Lys Glu Arg Leu Leu Asp Asp Arg His Asp Ala              20 25 30 Gly Leu Asp Ala Met Lys Asp Glu Glu Tyr Glu Gln Met Val Lys Glu          35 40 45 Leu Gln Glu Ile Arg Leu Glu Pro Gln Glu Val Pro Arg Gly Ser Glu      50 55 60 Pro Trp Lys Gln Gln Leu Thr Glu Asp Gly Asp Ser Phe Leu His Leu  65 70 75 80 Ala Ile Ile His Glu Glu Lys Ala Leu Thr Met Glu Val Ile Arg Gln                  85 90 95 Val Lys Gly Asp Leu Ala Phe Leu Asn Phe Gln Asn Asn Leu Gln Gln             100 105 110 Thr Pro Leu His Leu Ala Val Ile Thr Asn Gln Pro Glu Ile Ala Glu         115 120 125 Ala Leu Leu Gly Ala Gly Cys Asp Pro Glu Leu Arg Asp Phe Arg Gly     130 135 140 Asn Thr Pro Leu His Leu Ala Cys Glu Gln Gly Cys Leu Ala Ser Val 145 150 155 160 Gly Val Leu Thr Gln Ser Cys Thr Thr Pro His Leu His Ser Ile Leu                 165 170 175 Lys Ala Thr Asn Tyr Asn Gly His Thr Cys Leu His Leu Ala Ser Ile             180 185 190 His Gly Tyr Leu Gly Ile Val Glu Leu Leu Val Ser Leu Gly Ala Asp         195 200 205 Val Asn Ala Gln Glu Pro Cys Asn Gly Arg Thr Ala Leu His Leu Ala     210 215 220 Val Asp Leu Gln Asn Pro Asp Leu Val Ser Leu Leu Leu Lys Cys Gly 225 230 235 240 Ala Asp Val Asn Arg Val Thr Tyr Gln Gly Tyr Ser Pro Tyr Gln Leu                 245 250 255 Thr Trp Gly Arg Pro Ser Thr Arg Ile Gln Gln Gln Leu Gly Gln Leu             260 265 270 Thr Leu Glu Asn Leu Gln Met Leu Pro Glu Ser Glu Asp Glu Glu Ser         275 280 285 Tyr Asp Thr Glu Ser Glu Phe Thr Glu Phe Thr Glu Asp Glu Leu Pro     290 295 300 Tyr Asp Asp Cys Val Phe Gly Gly Gln Arg Leu Thr Leu 305 310 315

Claims (16)

a) obtaining a transformed cell by introducing into the exosome-producing cell a polynucleotide encoding a fusion protein in which a protein that inhibits exosome-specific markers and NF-κB is bound; And
and b) culturing the transformed cells. The method for producing a large amount of exosome in which an NF-κB inhibiting protein is present in an exosome.
The method according to claim 1, further comprising, when the NF-kB inhibiting protein is attached to the exosome membrane, separating the NF-kB inhibiting protein from the exosomal membrane in step b) A method for producing a large amount of exosome existing in an exosome characterized by the following.
3. The method according to claim 1 or 2, wherein the protein that inhibits NF-kB is any one or more selected from the group consisting of proteins represented by any one of SEQ ID NOS: 1 to 5, A method for mass production.
The method of claim 1 or 2, wherein the exosome-producing cell is selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, Of the total amount of the exosome.
The method according to claim 1, wherein the exosome-specific marker is CD9, CD63, CD81 or CD82.
a) a polynucleotide encoding a fusion protein (first fusion protein) in which the exosome-specific marker and the first photo-specific binding protein are bound to the exosome-producing cell, and a polynucleotide that binds to the first photo- Introducing a polynucleotide encoding a fusion protein (second fusion protein) in which a second photo-specific binding protein capable of binding to NF-κB is bound to a protein that inhibits NF-κB;
b) irradiating the exosome-producing cell with light capable of inducing binding of the first photo-specific binding protein and the second photo-specific binding protein; And
and c) stopping the irradiation of the exosome after the exosome is produced in the exosome-producing cell, and then stopping the irradiation of the exosome.
7. The method of claim 6, wherein the first photo-specific binding protein is selected from the group consisting of cryptochrome-interacting basichelix-loop-helix protein (CIBN), N-terminal domain of CIB, phytochrome B, phytochrome interacting factor (PIF) , FKF1 (Flavinbinding, Kelch repeat, Fbox 1), GIGANTEA, CRY (chryptochrome) and PHR (phytolyase homolgous region).
7. The method of claim 6, wherein when the first photo-specific binding protein is CIB or CIBN, the second photo-specific binding protein is CRY or PHR, and the second photo- Wherein the binding of the protein is carried out by irradiating light having a wavelength of 460 to 490 nm.
7. The method of claim 6, wherein when the first photo-specific binding protein is PhyB, the second photo-specific binding protein is PIF, and the binding of the first photo- specific binding protein and the second photo- And irradiating light having a wavelength of 600 to 650 nm. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
7. The method of claim 6, wherein when the first photo-specific binding protein is GIGANTEA, the second photo-specific binding protein is FKF1, and the binding of the first photo- specific binding protein and the second photo- Characterized in that the method is carried out by irradiating light having a wavelength of 460 to 490 nm.
An exosome comprising an internally contained protein that inhibits NF-kB, produced using the method of any one of claims 1, 2 or 6.
A method for delivering a protein that inhibits NF-κB to a cytoplasm, characterized by using the exosome of claim 11.
11. A pharmaceutical composition for the prevention and treatment of inflammatory diseases comprising the exosome of claim 11 as an active ingredient.
14. The method of claim 13, wherein the inflammatory disease is selected from the group consisting of allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, inflammatory bowel disease, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, acute and chronic inflammatory diseases, sepsis, , Ulcerative colitis, and Crohns disease. &Lt; Desc / Clms Page number 24 &gt; 6. A pharmaceutical composition for the prevention and treatment of inflammatory diseases.
(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in which a first light-specific binding protein and an exosome-specific marker are bound; And
(b) a polynucleotide capable of introducing a polynucleotide encoding a protein that inhibits NF-κB and a polynucleotide encoding a second photo-specific binding protein capable of binding to the first photo-specific binding protein, And a second expression vector comprising the expression vector.
[14] The method of claim 13, wherein the second expression vector is one in which a fusion protein (second fusion protein) in which the second photo-specific binding protein and a protein that inhibits NF-κB are fused is expressed in an exosome-producing cell Vector for producing exosome.

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