WO2016178532A1

WO2016178532A1 - Production method for exosome comprising target protein, and method for transferring target protein into cytoplasm by using exosome produced by means of the production method

Info

Publication number: WO2016178532A1
Application number: PCT/KR2016/004750
Authority: WO
Inventors: 최철희; 임남빈; 허원도; 류승욱; 최호준; 최경선
Original assignee: 한국과학기술원
Priority date: 2015-05-04
Filing date: 2016-05-04
Publication date: 2016-11-10

Abstract

The present invention relates to a method for producing a large volume of an exosome comprising a target protein, to an exosome-producing vector that can be used in the exosome production, to an exosome comprising a target protein produced by means of the method, and to a method for transferring the target protein into cytoplasm by using the exosome which is produced. When using the production method for an exosome comprising the target protein provided by the present invention, the exosome comprising the target protein can be produced in high yield, and thus the present invention can be widely used in disease treatment using the exosome.

Description

【Specification】

[Name of invention]

Method for producing exosomes containing the target protein and delivery of the target protein to the cytoplasm using the exosomes prepared by the above method:

Technical Field

In addition, the present invention relates to a method for producing an exosome containing a target protein using a photospecific binding protein, and to a method for delivering a target protein to the cytoplasm using the exosome prepared by the method.

[Technique to become background of invention]

The human body is composed of about 200 trillion cells and maintains life by controlling physiological activity by the action of various proteins in the cells.

The cells are surrounded by a double membrane structured membrane composed of phospholipids, and the membrane prevents the entry of foreign substances into the cells. Most protein therapeutics developed so far do not enter the cytoplasm through the cell membrane, and have a physiological effect by acting outside the cell or by receptors on the cell membrane to transmit signals into the cell.

There are many proteins in the cytoplasm, and they interact with each other to regulate physiological activity. If the protein therapeutics can be put directly into the cell, ie into the cytoplasm, it will be able to control the activity of the cell more effectively. Recently, research into a method of directly introducing a target protein through a cell membrane into a cell has been actively conducted. Protein Transduct Ion Domains (PTDs) and recombinant proteins of the protein of interest can be made and administered through the cell membrane to enter the cytoplasm (Fig. 1). The membrane transmembrane domain (PTD) includes HIV-1 TAT, HSV VP22, Antp, dfTAT, Hph-1, and the like, and the fusion protein combining the target protein is produced in recombinant protein form and needs to be separated. ， In this process, the protein refolding is not properly done, the activity is reduced, the protein is non-specific

One

Alternative Site (Article 26) There is a problem that the delivery, the risk of causing an immune response in vivo, high cost, low yield.

The desired protein combined with various nanoparticles can enter the cytoplasm through the cell membrane by endocytosis (FIG. 2). Nanoparticles include Gold NP, Liposome NP, Magnet icNP, Polymeric NP, and the degradation of the conjugate of these and the protein of interest occurs mostly in the lysosomes in the cell: the target protein is degraded inside the lysosome and loses its activity. Discarded or cytosolic, it is difficult to separate the target protein and nanoparticles separately, and the toxicity of the nanoparticles can be a problem. Exosomes refer to small vesicles of 50-200 nm size that are secreted out of cells with proteins, DNA, RNA, etc. for intercellular signaling. ^'

Exosomes were originally discovered in the process of leaving and removing hemoglobin in red blood cells by releasing and removing intracellular proteins at the last stage of maturation of red blood cells. These exosomes are separated directly from the plasma membrane in electron microscopic studies. Rather, it was observed to be released and secreted out of the cell, originating in a specific compartment within the cell called multiivesicular bodies MVBs. That is, when fusion occurs between the polycystic body and the plasma membrane, such vesicles are released into the extracellular environment, which is called axosome (Fig. 3).

It is not clear what molecular mechanism these exosomes are made of, but various kinds of immune cells and stem cells, including B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, etc. Tumor cells are also known to produce and secrete exosomes in the living state.

Exosomes contain a variety of proteins, DNA, RNA, etc. in the cell. Substances contained in these exosomes and secreted out of the cell are re-introduced into other cells by fusion or endocytosis with the cell membrane, thereby serving as a communication between cells. In addition, the material contained in such exosomes secreted out of the cell can be used to diagnose specific diseases.

Exosome contains several kinds of microRNAs, the presence or absence of them

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Alternative Site (Article 26) And a method for diagnosing a disease by detecting abundance (see KR 10-2010-0127768A). In particular, international publication W02009-015357A detects exosomes present in cancer-derived samples (blood, saliva, tears, etc.), measures the amount of mi croRNA changes, and increases or decreases compared to the control group. A method of predicting and diagnosing is disclosed. In particular, exosomes obtained from patients with specific diseases (pulmonary diseases) have been analyzed to specifically disclose the association between specific mi croRNA and lung diseases. In addition, in addition to lung diseases, a method for diagnosing kidney disease using a protein contained in exosomes is being studied.

Axosomes also contain antigens. Meanwhile, in antigen presenting cells (ant igen present cel l, APC), antigenic peptides are loaded into MHCOnajor hi stocompat ibi li ty complex (IIC) class II molecules in intracellular compartments including polycystic bodies. The resulting axosomes also have antigen peptide MHC class II complexes. Thus, exosomes can present antigenic peptides to CD4 + T lymphocytes as transporters of immunogens, thereby inducing immune responses such as proliferation of T lymphocytes. In addition, exosomes are enriched with molecules capable of stimulating immune reactions, such as C-Class I and heat shock proteins (HSPs), which can be used to treat autoimmune di sease or tumors. It can be used for the purpose of immune augmentation or reduction.

In addition, the exosomes containing the target protein therein can be used for the treatment of various diseases in vivo. For example, an exosome containing a protein or siRNA having an anticancer effect as a target protein can be prepared, and treated with cancer cells to be used for cancer treatment (FIG. 4).

As such, exosomes containing the target protein can be used for the treatment of diseases. This requires efficient production of exosomes containing the protein of interest. Korean Laid-Open Patent No. 2004-0015508 discloses a method for preparing axosome comprising a specific antigen. By introducing a gene for a specific antigen into a cell line, a protein of the introduced gene is stably expressed in the cell line; a method of releasing out of a cell through an exosome and a method of using such an exosome as a vaccine are disclosed. .

However, because the exosomes are naturally formed in the cell, S,

3

Alternative Site (Article 26) Which encodes the protein of interest in the cell that produces the exosomes. Even if the gene is introduced, there is a problem that the likelihood of producing a exosome containing the expressed protein therein is very low.

Accordingly, the present inventors have conducted research to develop a method for more efficiently preparing axosomes containing a target protein. As a result, the present inventors have expressed a fusion protein obtained by fusing an exosome-specific marker and a target protein in a cell that produces a large amount of axosome. , It was confirmed that the exosomes containing the target protein can be efficiently produced (Fig. 5).

In addition, the method is that the target protein is attached to the membrane of the exosomes, ^{° °} cells in which a large amount of exosomeol fusion protein fusion of each of the exosome-specific marker and each of the photospecific binding protein pairs to the target protein When expressing and binding the respective fusion proteins by irradiation with light, these binding proteins are introduced into the exosomes by exosome-specific markers. It was confirmed that the fusion protein of the host binding protein can be isolated to effectively prepare the exosomes containing the target protein in the free state therein (FIG. 6).

In addition, by using the exosomes contained within the target protein, confirming that the target protein is delivered to the cytoplasm of the target cell, by efficiently controlling the intracellular signal in the cytoplasm (Intracel l s ignal ing) to control the disease A method of treatment is presented.

Detailed description of the invention

[Technical problem]

It is an object of the present invention to provide a method for mass production of exosomes comprising a fusion protein of an exosome specific marker with a protein of interest. ^'

It is another object of the present invention to provide a method for mass production of exosomes comprising a target protein isolated from an exosome membrane using photospecific binding protein pairs.

Another object of the present invention is to provide a exosome gymnastics vector that can be used in the production of the exosomes.

Another object of the present invention is to use the exosomes in the cells ^' , quality: the target protein

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Alternative Site (Article 26) It is about a method of inflow.

Technical Solution

The present invention relates to a method for efficiently producing large amounts of exosomes containing the protein of interest.

The present inventors have been paying attention to exosome-specific markers (CD9, CD63, CD81 or CD82) while performing various facets in order to develop a method for more efficiently preparing the exosomes in which the target protein is contained therein. Since the exosome-specific markers have in common that they are four membrane-penetrating membrane proteins belonging to the tetraspanin family, when the target protein is bound to the membrane protein of the exosomes, the exosome-specific markers can be easily contained in the exosomes. Predicted.

As such, by expressing abundantly specific exosomes on the exosome membrane and expressing a fusion protein of axosome-specific markers and target proteins in the cells that make a large amount of exosomes, a large amount of exosomes containing the desired protein can be produced. . ^'

Specifically, the method for producing an exosome containing the target protein of the present invention is characterized by introducing and expressing a polynucleotide encoding a fusion protein in which an exosome-specific marker and a target protein are bound in an exosome-producing cell.

In the exosomes produced at this time, the target protein is fused with an exosome-specific marker embedded in the exosome 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 this problem, as a result of various studies, using a photospecific binding protein, by temporarily binding the target protein with the marker protein, a technique for producing an exosome containing the target protein therein Developed. For example, the photospecific binding proteins CIBN and CRY2 can be used. Specifically, the CIBN is expressed in a fused form with CD9, which is one of the marker proteins, and the CRY2 is in a fused form with a target protein. Genes encoding these fusion proteins to be expressed were introduced into exosome producing cells. The CIBN-CD9 fusion protein expressed in the exosome-producing cells is included in the exochom due to CD9. At this time, when the cells are irradiated with blue light LED light, the replacement paper (rule 26) Since the CRY2 domain of the protein-CRY2 fusion protein expressed in the exosome-producing cells is bound to the CIBN domain fused to CD9, a reversible induced fusion protein of the protein-CRY2-CIBN-CD9 form is formed. Fusion proteins are included inside the exosomes due to CD9. After the exosomes containing the target protein are produced in this way, if the blue light LED is no longer irradiated, CIBN-CRY2 binding is released, and the target protein is not bound to the exosomes cell membrane. Since it is included in, it can be prepared as a result of the exosomes containing the target protein (Figs. 5 to 10).

The exosomes thus prepared may exhibit completely different effects from the exosomes including the conventional target material. Conventional exosomes were expressed in a form fused to an exosome-specific marker in order to capture the target protein inside the exosome, but the target protein was collected in the exosome, but attached to the mexosome membrane. because of it not being separated from the exo sommak, only when fused to a cell membrane of the exo somyi target cells, and wherein the desired protein can be delivered to the target cells, thus even after the convergence to a target cell, fused to a solution sosom ^film: exo because it exists in a form bound to cotton, wherein the target protein is very low hwakreul exhibit its effect in the target cell was boundaries hinge ^i. However, since the exosomes provided in the present invention are trapped inside the exosomes in a state in which the target protein is not bound to the membrane, when the exosomes are endocytosi s by target cells and enter the cytoplasm, they are attached to the exosomes membrane. When the exosomes are decomposed, the target protein can be delivered to the cytoplasm of the target cell, and since the target protein can be freely moved within the cytoplasm of the target cell, the target protein can sufficiently exhibit the physiological activity in the target cytoplasm. There is this (Figure 11).

In addition, since the binding level of the target protein bound to the marker protein is changed according to the light intensity irradiated upon light irradiation, there is an advantage that the content of the target protein collected in the exosomes can be adjusted when the light intensity is adjusted. .

Therefore, a method for producing an exosome containing a target protein using a photospecific binding protein is not known at all, and was first developed by the present inventors.

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Alternative Site (Article 26) Specifically, the method for producing an exosome comprising the protein of interest of the present invention is (a) a fusion protein in the form of binding an exosome-specific marker and a crab 1 photospecific binding protein to an exosome-producing cell (first fusion protein ), A polynucleotide encoding the nucleotide and a second photospecific binding protein capable of binding the first photospecific binding protein and a polynucleotide ¾ encoding a fusion protein (second fusion protein) in a form in which the target protein is bound. Doing; (b) irradiating the axosome-producing cells with light capable of causing the binding of the giant U photospecific binding protein to the second photospecific binding protein; And (c) after the exosomes are produced in the exosome-producing cells, stopping the irradiation of the light.

As used herein, the term "exosome" refers to a small vesicle of plasma membrane structure that originates in a specific compartment in a cell called mult ivesicular bodies (MVBs) and is released or secreted out of the cell.

In the present invention, the exosomes may serve as a carrier for transporting the target protein to the target cells or tissues of the target protein, including the target protein well therein, the target protein carried by the exosome is a target It can be used to treat a particular disease or diagnose a particular disease by acting on a cell or tissue.

The infant "exosome producing cell" of this invention means the cell which can produce the said exosome.

In the present invention, the exosome-producing cells are not particularly limited thereto, but as an example, B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, tumor cells, etc. Can be. For example, in the embodiment of the present invention, HEK293T cells, which are a kind of immortalized cell line (i 匪 ortal i zed cel l ine), were used as the exosome-producing cells.

The term "lixosome specific marker" of the present invention means a protein abundantly present in the membrane of exosomes. In the present invention, the exosome-specific marker is not particularly limited to, alternative paper (rule 26) As one example, it may be CD9, CD63, CD81, CD82, or 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 four transmembrane proteins. When the target protein is bound to the membrane protein of the exosomes, the target protein is easily present inside the exosomes.

The term “photospecific binding protein” of the present invention is also referred to as a photoinduced heterodimer-forming protein or a photoinduced homodimer-forming protein. When light of a specific wavelength is irradiated, it may bind to different proteins to form a heterodimer. It refers to a protein that can be combined with other proteins of the same type or to form a homodimer.

In the present invention, the photospecific binding protein is not particularly limited thereto, but as an example, it may be a photoinduced heterodimer-forming protein, and as another example, CIB (cryptochrome ᅳ interacting bas i c-he 1 i χ-1 oop-he 1 ix protein, CIBN (N—terminal domain of CIB), PhyB (phytochrome B), PIF (phytochrome interacting factor), FKFKFlavinbinding, Kelch repeat, F 一 box 1), GIGANTEA, CRY or (chryptochrome) PHR (phyto lyase homolgous region).

In particular, in the case of forming a release dimer, the two light specific binding proteins there (the first optical-specific binding protein and the second optical-specific binding protein) is to be used can be ^", the first light-specific binding protein is a CIB or In the case of CIBN, the second photospecific binding protein for fish may be CRY or PHR, and if the first photospecific binding protein is PhyB, the second photospecific binding protein for this may be PIF. And, if the U U-specific binding protein is GIGANTEA, the second photospecific binding protein for this may be FKF1.

For example, in the embodiment of the present invention, CIBN was used as the first photospecific binding protein, CRY2 was used as the second photospecific binding protein, and the wavelength of light used was set to 460 to 490 nm indicating blue light. And, the light intensity was set to 20 to 50.

Meanwhile, the marker protein may be fused together to confirm the expression and intracellular location of the C1 fusion protein to which the exosome-specific marker and the first photospecific binding protein are bound. For example, in the embodiment of the present invention, EGFP, which is a fluorescent protein, is inserted into a first fusion protein in which CIBN and CD9 or GIGANTEA and CD9 are combined.

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Alternative Site (Article 26) By expressing, the expression, expression level and intracellular location of the first fusion protein were determined. As used herein, the term "target protein" refers to a protein expressed in a fused form with the second photospecific binding protein to be included in the axosome.

In the present invention, the target protein is not particularly limited as it can be expressed in cells and transported through exosomes, but may be a protein for treating diseases, a disease: a diagnostic protein, and the like as an example. For example, mCherry, which fluoresces, was used as the target protein in Examples of the present invention. As used herein, the term "culture" refers to a method of growing cells or microorganisms under suitable artificial conditions.

In the present invention, the transformant is cultured for 1 to 3 days, then replaced with a medium containing no fetal bovine serum and further cultured for 2 to 5 days.

In the present invention, the method of culturing the transformant may be performed using a method well known in the art. ^'

The medium refers to a known medium used in animal cell culture, and is a group of commercially available serum free media, protein free media, and chemically defined indium. Can be selected from.

The serum-free medium is a medium from which the bovine serum components used for culturing animal cells are removed, and SFM4CH0 (HyClone), EX—Cel KJHR Biosci- ence, etc. are preferred, and insulin type growth factor K lnsul in l ike growth factor I, IGF-I), ethanol amine, Ferric chloride, and phosphatidylcholine may be added, but are not limited thereto.

The protein-free medium is an animal cell culture in which a serum-free component is removed from an animal-free protein, in particular, an animal cell culture in which a polymer protein having a molecular weight of lOkDa or more is removed 'is selected from ProCHO (Lonza) and PF-CHO (HyClone). It may be, but is not limited thereto.

The chemically defined medium is a medium for animal cell culture which is free of components of animal milk sfl, and all components of the medium have a known chemical structure.

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Alternative Site (Article 26) CDM4CH0 (HyClone), PowerCH02CD (Lonza), CD-opt iCH0 (Li Technologies), etc. ⁰ may be selected, but is not limited thereto. The term "first fusion protein" of the present invention means a fusion protein in a form in which the exosome-specific marker and the first photospecific binding protein are bound to each other.

In the present invention, the arrangement sequence of the exosome-specific marker and the first photospecific binding protein contained in the first fusion protein is the first fusion protein in the direction of the exosomes in the exosomes in the axosome-producing cells As long as the specific binding protein can be expressed so as to be located, it is not particularly limited thereto, but as an example, the C-terminus of the exosome-specific marker may be composed of the N-terminus of the first photospecific binding protein. Can be. In addition, the exosome-specific marker and the system 1 photospecific binding protein constituting the first fusion protein may be directly connected to each other or may be linked through a linker. The linker may use a peptide linker composed of amino acids, although not particularly limited thereto, as long as the first fusion protein can be expressed such that the first photospecific binding protein is located inwardly of the exosome in an exosome producing cell. It is possible to use a more flexible peptide linker. The peptide linker may be expressed by linking the nucleic acid encoding the linker in frame between the nucleic acids encoding the respective domains in an operably linked expression vector. The term "second fusion protein" of the present invention means a fusion protein in a form in which the second photospecific binding protein and the target protein are bound.

In the present invention, the sequence of the second photospecific binding protein and the target protein included in the second fusion protein, the first photospecific of the first fusion protein in the axosome-producing cells As long as it can bind to the binding protein site and be located inside the exosome, the present invention is not particularly limited thereto. As one example, the C-terminus of the second photospecific binding protein is envisioned in the form of the N-terminus of the target protein. Can be. In addition, the second photospecific binding protein and the target protein constituting the second fusion protein may be directly connected to each other, or may be linked through a linker.

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Alternative Site (Article 26) The linker is a peptide consisting of amino acids, although not particularly limited thereto, as long as the second fusion protein can be located inside the exosome by binding to the nearly U photospecific binding protein site of the first fusion protein in axosome-producing cells. Linkers may be used, and more preferably, a flexible peptide linker may be used. The peptide linker may be expressed by linking the nucleic acid encoding the linker in frame between the nucleic acids encoding the respective domains in an operably linked expression vector. In addition, the fusion protein may include a polypeptide having a sequence in which at least one amino acid residue is different from the amino acid sequence of the wild type of each domain included therein. Amino acid exchanges in proteins and polypeptides that do not alter the activity of the molecule as a whole are known in the art. The most commonly occurring exchanges include the amino acid residues Ala / Ser, Val / I le, Asp / Glu, Thr / Ser, Ala / Gly, Al a / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / I le, Leu / Val, Ala / Glu, Asp / Gly. In addition, the protein may include a protein having increased structural stability or increased protein activity against heat, pH, etc., due to a mutation or modification on the amino acid sequence.

Finally, the fusion protein or polypeptide of each domain constituting the fusion protein may be prepared by a chemical peptide synthesis method known in the art, or the gene encoding the domain may be amplified or known by PCR (polymerase chain react ion). After synthesis by the conventional method can be prepared by cloning the expression vector expression. On the other hand, each of the fusion proteins can be expressed in the exosome-producing cells by introducing the hoolinucleotides encoding them into the exosome-producing cells, which is known to those skilled in the art as a method for introducing the polynucleotide into the exosome-producing cells. The method may be used, for example, a method of introducing using an expression vector may be used.

As used herein, the term “expression vector” refers to a gene construct that is a recombinant vector capable of expressing a peptide of interest in a host cell of interest, and which includes essential regulatory elements operably linked to express the gene insert. The expression vector is

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Alternative Site (Article 26) Initiation codon, termination codon, promoters, comprising the expression control elements, such as the operator, the start codon and stop codon are generally the ^polypeptide: is considered to be part of a nucleotide sequence encoding, must be in the object when a genetic construct is administered It should be functional and be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

As used herein, the term “operably l inked” refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. Means. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the coding sequence. Operative linkage with expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can employ enzymes commonly 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. Specific initiation signals may also be required for efficient translation of inserted nucleic acid sequences. These signals include ATG start codons and contiguous sequences. In some cases, an exogenous translational control signal must be provided that can include an ATG start codon. These exogenous translational, regulatory signals and initiation codons can be various natural and synthetic sources. Expression efficiency can be increased by the introduction of appropriate transcriptional or translation enhancing factors. According to a preferred embodiment of the present invention, the expression vector may be expressed by binding a tag capable of confirming whether the target protein is inserted into the exosome to the target protein. Since the tag is for confirming the presence of the target protein, the target protein can be bound to the opposite site combined with the second photospecific binding protein. For example, a fluorescent protein such as a red fluorescent protein or a green fluorescent protein can be bound. Can be used as a tag to bind to the C-terminal region of the protein of interest.

This designed protein is expressed in exosome-producing cells, and the exosomes

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Alternative Site (Article 26) After production, it is possible to confirm whether the exosomes contain the target protein by checking whether the fluorescent protein tag is detected in the exosomes. The term "light" of the present invention means light irradiated to temporarily bind a first photospecific binding protein and a second photospecific binding protein expressed in exosome-producing cells.

As described above, in the exosome-producing cells, the first photospecific binding protein is expressed in the form of a first fusion protein with an exosome-specific marker, and the second photospecific binding protein together with the target protein is a second fusion protein. When the exosome-producing cells are irradiated with light necessary for the binding of the first photospecific binding protein and the second photospecific binding protein to the exosome-producing cells, the first photospecific binding protein and the second photospecific light are expressed. The binding protein is combined to result in a temporary formation of a fusion protein complex having the shape of an exosome, a specific marker-first photospecific binding protein-second photospecific binding protein-target protein, wherein the exo When exosomes are produced in som-producing cells, the exosome-specific markers allow the protein of interest to be linked to exosomes. In this case, the target protein is present in the exosomes, and when the irradiation of light stops after the exosomes are produced, the first photospecific binding protein and the second photospecific binding protein 'is separated, and accordingly, The desired protein present in the exosomes is released to the outside in the form contained in the exosomes upon release of the exosomes. In addition, the light is preferably irradiated intermittently rather than continuously irradiated so that the target protein can be introduced into the exosomes more effectively. That is, in the case of irradiating light intermittently, since the binding and separation of the crab 1 photospecific binding protein and the second photospecific binding protein are repeated, the probability of the target protein being introduced into the exosome can be improved. .

On the other hand, since the wavelength of the light that induces the binding of the first photospecific binding protein and the second photospecific binding protein depends on the type of the first photospecific binding protein and the second photospecific binding protein, those skilled in the art As known to the present invention, the wavelength of 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 the PhyB and PIF are combined, the light having a wavelength of 650 nm is irradiated for 10 minutes. The light having the wavelength of 750 nm is irradiated for 5 minutes.

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Alternative Site (Article 26) Dissociate from each other; When FKF1 and GIGANTEA are combined, light with a wavelength of 460 nm can be irradiated for 30 minutes. In the embodiment of the present invention was irradiated with light having a wavelength of 460 ~ 490nm to induce the binding of CIBN and CRY2.

According to an embodiment of the present invention, as a result of expressing the fusion protein of CRY2 and mCherry and the fusion protein of CIB and CD9 in HEK293T cells, an immortal cell line that produces a large number of exosomes, the distribution of mCherry protein uniformly spread in the cytoplasm When exposed to blue light, it could be observed that the cell membrane, endosomes-like structures, etc. (Fig. 7). Similar results were observed when FKF1 and mCherry fusion proteins and GIGANTEA and CD9 fusion proteins were expressed in HEK293T cells (FIG. 12). In addition, by expressing the fusion protein of CRY2 and mCherry and the fusion protein of CIBN and CD9 to HEK293T, and control the blue light intensity, when the irradiation of 20 to 50, the highest mCherry protein captured in the exo It was confirmed that the level (Fig. 9), the exosomes produced from the cells was treated with a concentration of about 25 C ^ g / m to ΗΊΊ080 cells, which are heterologous cells, did not show any particular cytotoxicity to Η Ί080 cells , It was confirmed that mCherry protein is delivered to the Η Ί080 cells and the cytoplasm (Fig. 10).

In addition, in order to compare the efficiency of introduction of the target protein in the exosomes of the present invention and the efficiency of delivery of the exosomes to the target cells with conventional methods, using the XPACK vector in the conventional method, the CRY2 and mCherry protein of the present invention After introducing the expression vector of the fusion protein in the form of fusion protein and CIBN and CD9 protein into HEK293T, and comparing the target protein production in dexosome, the introduction efficiency was remarkably improved when the method of the present invention was used. This was confirmed to be high (FIG. 15). In addition, when the exosomes isolated from the exosome-producing cells were treated with the target cells (HeLa), and the degree of expression of the target protein was compared, when the exosomes separated by the method of the present invention were used, It was confirmed that the expression was the highest (FIG. 16). According to another aspect of the present invention, (a) a first expression vector comprising a polynucleotide encoding a fusion protein (a first fusion protein) in a form in which a exosome-specific marker and a first photospecific binding protein are bound; And (b) a mult icloning site into which a polynucleotide encoding a protein of interest can be introduced and the first

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Alternative Site (Article 26) Provided is a vector for producing an exosome, comprising a second expression vector comprising a polynucleotide encoding a second photospecific binding protein capable of binding to a photospecific binding protein. In the exosome preparation vector provided by the present invention, the exosome specific marker, the first photospecific ^' binding protein, the exosome producing cell and the second photospecific binding protein are the same as described above. The term "transformed cell for exosome production" of the present invention, encoding a fusion protein (low U fusion protein) of the form in which the exosome-specific marker and the first photospecific binding protein is bound to the exosome-producing cells Polynucleotide is introduced, and means: axosomal producing cells capable of expressing the first fusion protein. In the present invention, the second expression vector includes a polynucleotide encoding the second photospecific binding protein, and may be configured to include a polyclonal region adjacent to the second expression vector. When a polynucleotide encoding a target protein is introduced into the polyclonal region of, the second photospecific binding protein and the target protein may be expressed in a fused form (second fusion protein). The vector for producing exosomes provided in the present invention is not only the transformed cells and expression vector for producing exosomes, but also the introduction of the expression vector, culture of transformed cells for exosomes production, produced from transformed cells for exosomes production. One or more other component compositions, solutions or devices suitable for isolating and purifying exosomes may be included. For example, it may further include a suitable buffer for the introduction of the expression vector, a medium and a container required for culturing the transformed cells for producing exosomes. In another aspect, the present invention is prepared by the above method, to provide an axosome containing the target protein therein.

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Alternative Site (Article 26) Exosome prepared by the above-described method includes a fusion protein (first fusion protein) in the form of a combination of the exosome-specific marker and the first photospecific binding protein in the plasma membrane constituting it, the inside of the exosome Since the second photospecific binding protein capable of binding to the first photospecific binding protein and a fusion protein (second fusion protein) in a form in which the target protein is bound are included, the exosomes are transferred to cells in the target tissue. Upon treatment, the second fusion protein contained within the axosome can be delivered to the cytoplasm in the target tissue through the fusion oligo of the plasma membrane. Axosomes containing the target protein therein can be used for the treatment of various diseases in vivo. For example, if an exosome containing a proteinaceous high molecule (for example, an antibody, etc.) exhibiting an anticancer effect as a target protein is produced, and treated with cancer cells, the exosomes may be used for bio-friendly chemotherapy than conventional liposomes. Can be.

【Effects of the Invention】

By using the method for producing an exosome containing the target protein provided in the present invention, it is possible to prepare a exosome containing the target protein in a high yield, and also because the target protein is separated from the exosome membrane s May be widely used for the treatment of diseases using exosomes ᅳ

[Brief Description of Drawings]

1 is a diagram showing the cytoplasmic delivery of the target protein through the membrane transmembrane domain (PTD) and the recombinant protein of the target protein (Steven R. et al. Protein transduction: unrestricted del ivery into al 1 eel Is? Trends in Cell biology, 2000). FIG. 2 is a diagram illustrating a method for intracellular delivery of a target protein through endocytosis of a conjugate of nanoparticles and a target protein (Munish Chanana et al. Physicochemical properties of protein-coated gold nanopart icles in biological fluids and eel Is before and after proteolytic digestion.Angew.Chem.Int.Ed. 2013).

FIG. 3 is a diagram showing a process in which exosomes are released from cells and separated from intracellular multi vesicular bodies (MVBs) (Graca Raposo and Willem)

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Alternative Site (Article 26) Stoorvogel. Extracel lular vesi cles: Exosomes, mi crovesi cles, and fr iends. Cel l Biology 200 (4), 373-383, 2013).

4 is a diagram showing a process of treating cancer by delivering siRNA * in vivo through targeted exosomes (Alvarez-Ervi ti, L. et al. Del ivery of siRNA to the mouse brain by systemic inject ion of targeted exosomes Nature biotechnology 29, 341-345, 2011).

Figure 5 is a schematic diagram showing the manufacturing process of opto-genetically designed protein transport exosomes (protein-carrying exosomes, EXPLORs) according to the present invention.

6 is a view showing a process of separating the fusion protein of the target protein and photospecific binding protein in the exosomes when light irradiation of the EXPLORs according to the present invention is stopped.

FIG. 7 is a fluorescence photograph showing the intracellular location change of mCherry protein according to whether blue light is irradiated on transformants in which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells.

8 is a view showing an experimental process for obtaining EXPLORs according to the present invention. 9 is an immunoblot analysis photograph showing the result of measuring the change in the content of the target protein (mCherry protein) collected in the exosomes according to the intensity of blue light.

10 is an electron micrograph showing a result of confirming whether or not the target protein is introduced into the target cell after treating the exosome containing the target protein (mCherry) to the target cell (HT1080 cell), and the left side is treated with the exosome. Unmarked target cells and the right side show target cells treated with exosomes.

11 is a fluorescence photograph (a) showing the result of confirming whether the target protein is introduced into the target cell after treating the exosome containing the target protein (mCherry) to the target cell (HT1080 cells) and the exosome treatment Is a graph showing the result of comparing the percentage of dead cells induced by the (b).

FIG. 12 is a fluorescence photograph showing the intracellular location change of the mCherry protein according to whether blue light is irradiated in a transformant in which the GIGANTEA-EGFP-CD9 gene and mCherry-FKFlLOV are introduced into HEK293T cells.

13 is the expression level of Luci ferase-mCherry fusion protein using fluorescence imaging (a)

17 ^' Alternative Site (Article 26) And (b) confirming luciferase activity and number of molecules in producing cells.

Control: HEK293T cells that did not process anything;

OVER: HEK293T cells incorporating Luc i aser-mCherry-CRY2 only;

XP: HEK293T cells incorporating XPACK-Luci ferase-mCherry using XPACK (Systems Biosciences), a commercial vector made for exosome loading technology;

EXPL0R: HEK293T cells incorporating Luci ferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the method of the present invention.

14 is a diagram confirming luciferase activity measurement (a) and number of molecules (b) in the produced exosomes:

NEG: exosomes produced in HEK293T cells treated with nothing;

OVER: axosomes produced in HEK293T cells incorporating Luc i ferase-mCherry-CRY2;

XP: Exosome produced in HEK293T cells introduced with XPACK-Luci ferase-mCherry using XPACK (Systems Bioscences), a commercially available vector made for exosome loading technology;

EXPL0R: Exosomes produced from HEK293T cells incorporating Luci ferase-mCherry—CRY2 and CIBN-EGFP-CD9 according to the present invention;

ON: Axosome produced by incubating for 72 hours in blue light of 200 μ \ ν,

OFF: Exosomes produced after 72 hours of incubation in the absence of light.

Figure 15 is a diagram showing the loading efficiency (efef i c iency) of the target protein in the produced exosomes.

Figure 16 shows the efficiency of protein delivery to target cells (HeLa) using axosomes:

Control: exosomes produced from HEK293T cells treated with nothing;

OVER: exosomes produced in HEK293T cells incorporating Luci ferase-mCherry-CRY2;

XP: exosomes produced in HEK293T cells incorporating XPACK—Luc i ferase-mCherry using XPACK (Systems Biosciences), a commercial vector made for exosome loading technology;

EXPL0R: exosomes produced in HEK293T cells incorporating Luc i ferase-mCherry-CRY2 and CIBN-EGFP-CD9 according to the present invention;

ON: Exosome produced by incubating for 72 hours in blue light of 200,

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Alternative Site (Article 26) OFF: Exosomes produced after 72 hours of incubation in the absence of light.

FIG. 17 shows that Luci ferase-mCherry-CRY2 and CIBN-EGFP-CD9 are expressed at the same position in HEK293T cells.

[Best Mode for Carrying Out the Invention] _"

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Axosome

<1-1> Confirmation of the binding of CIBN and CRY2 for the preparation of axosome

PcDNA3 containing the CIBN-EGFP-CD9 gene in the absence of light. PcDNA3 containing 1 (+) vector and mCherry-CRY2 gene. 1 (+) vector was introduced into HEK293T cells, exosome producing cells, incubated for 24 hours, then replaced with medium containing fetal bovine serum and further incubated for 48 hours. After the incubation was completed, blue light having a wavelength of 460 to 490 nm was irradiated, and the position of red fluorescence appeared in mCherry before and after irradiating the blue light was confirmed through confocal microscopy (FIG. 7).

FIG. 7 is a fluorescence photograph showing the intracellular location change of mCherry protein according to whether blue light is irradiated in transformants in which CIBN-EGFP-CD9 gene and mCherry-CRY2 gene are introduced into HEK293T cells. As shown in FIG. 7, the mCherry protein is evenly distributed in the cytoplasm before the blue light that causes the binding of the photospecific binding protein CIBN and CRY2 is irradiated, but the mCherry protein is concentrated on the membrane after the blue light is irradiated. It was found that this appeared. The density of the mCherry protein was analyzed to be caused by the binding of CRY2 and CIBN, a photospecific binding protein.

<1-2> Confirmation of the binding of GIGANTEA and FKF1 for the preparation of axosome

PcDNA3 comprising the GIGANTEA-EGFP-CD9 gene. PcDNA3 comprising 1 (+) vector and mCherry-FKFlLOV gene. Intracellular exosomes were identified using a 1 (+) vector and using the same method as in Example <1-1>. (L0V in the FKF1L0V is

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Alternative Site (Article 26) In the abbreviation of ight-oxygen-volt age domain, FKF1 protein ^' indicates a domain that actually binds to other proteins by light, and thus FKF1 and FKF1L0V are used in the same manner as in Example <1-1>. As shown in FIG. 12, the mCherry protein is evenly distributed in the cytoplasm before the blue light irradiating the photospecific binding protein GIGANTEA and FKF1 is irradiated, but after the blue light is irradiated, the mCherry protein is concentrated in the membrane. It was confirmed that the phenomenon appeared (Fig. 12). Therefore, similar to the result of <1-1>, it was confirmed that the density of mCherry protein can be induced by the binding of GIGANTEA and FKF1 of the photospecific binding protein.

Example 2 Effect of Light Intensity on Exosomal and Axosome Production

HEK293T cells, which are exosome-producing cells, express expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry_CRY2 gene, respectively, under LED lamps emitting light of 460 nm wavelength at an intensity of 0, 5, 20, 50, or 200, respectively. It was introduced into, cultured for 24 hours, then replaced with medium containing fetal bovine serum and further cultured for 48 hours. After the incubation was completed, the culture solution was separated and centrifuged (3000xg, 15 minutes) to obtain a supernatant from which cell debris was removed. The obtained supernatant was mixed with 5 times the volume of the supernatant by adding ExoQui ck-TC Exosome Precipi tat ion Solut ion (System Biosciences, Mountain View, Cali fornia, USA) and centrifuged (1500xg, 30 minutes). To obtain the precipitated exosomes, and suspended in PBS was added to the obtained exosomes to obtain an exosomes suspension ᅳ The exosomes suspension was filtered through a 0.2 ^ ni filter using a syringe equipped with a 27-G needle A single sized xosome was obtained (FIG. 8). Thereafter, Exosome Lysate was made using Lysi s buf fer, and the amount of mCherry protein contained in exosomes was compared by immunoblot analysis (FIG. 9).

9 is an immunoblot analysis photograph showing the result of measuring the change in the content of the target protein (mCherry protein) collected in the exosomes according to the intensity of blue light. As shown in FIG. 9, when irradiated with blue light at an intensity of 20 to 50 μ \ ν, it was confirmed that the content of the target protein (mCherry protein) collected in the exosome showed the maximum value. From the above results, by adjusting the intensity of light irradiated upon binding of the photospecific binding protein,

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Alternative Site (Article 26) It was found that the content of the target protein trapped inside the axosome can be controlled.

Example 3 Treatment Effect of Axosome

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene, respectively, was introduced into HEK293T cells, which are axosome-producing cells, under LED lamps emitting light of 460 nm wavelength at an intensity of 50. The exosomes were extracted by the 2-method. Then, the extracted exosomes in a concentration of 250 in ΗΊΊ080 cells

Treatment was done for 24 hours. Then, the HT1080 cells were fixed by adding 0.1 M phosphate buffer (pH 7.4) containing 4% PFA and 0.01% GA, and attached onto 10% gelatin gel. Cells attached on gelatin gel were excised for one day with liquid nitrogen, and slices cut to 45 nm thickness using cryoul tramicrotome at -120 ^° C. Subsequently, the flakes were immunostained using anti-mCherry antibody and Protein A-gold, and the mCherry protein was observed through Tecnai G2 Spi rit Twin TEM (FIG. 10). .

10 is an electron micrograph showing a result of confirming whether or not the target protein is introduced into the target cell after treating the exosome containing the target protein (mCherry) to the target cell (HT1080 cell), and the left side is treated with the exosome. Unmarked target cells and the right side show target cells treated with exosomes. As shown in FIG. 10, when the exosome of the present invention was treated to target cells, the target protein was confirmed to be delivered into the target cells.

Example 4 Analysis of Axosomes Containing Target Proteins

Each of the expression vectors containing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene, respectively, was introduced into HEK293T cells, which are exosome-producing cells, under LED lamps irradiating light of 460 nm wavelength at an intensity of 50. Exosome was extracted by the method of 2. Subsequently, the extracted exosomes were treated in ΗΊΊ080 cells at a concentration of 250 / ^ for 24 hours. Then, the red fluorescence of the mCherry protein was confirmed by fluorescence microscopy, and the ratio of the dead cells of the exosome-treated and untreated cells was compared by the LDH cel l death assay (FIG. 11).

11 shows target cells (HT1080) containing exosomes containing the target protein (mCherry).

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Alternative Site (Article 26) Cells), and a graph showing the result of comparing the ratio of the fluorescence photograms (a) showing the result of introducing the target protein into the target cells and the percentage of dead cells induced by the exosome treatment. . As shown in FIG. 11, it was confirmed that apoptosis was not induced by the treatment of exosomes.

<Example 5> Production of axon and comparison of introduction efficiency of the target protein in the produced axon <5-1> Confirmation of axon production efficiency

In order to confirm the expression of the target protein in exosome-producing cells in order to compare the degree of introduction of the target protein in the exosomes produced and produced exosomes of the present invention, luciferase activity (luci ferase act ivi ty) measurement experiment This was done.

Conventionally, XPACK-Luci ferase-mCherry was introduced into HEK293T cells using XPACK (Systems Biosciences), a commercial vector made for axosome loading technology (XP), and Luci ferase-mCherry using the method of the present invention. -CRY2 and CIBN-EGFP-CD9 were introduced into HEK293T cells (EXPLOR), and then intracellular luciferase activity was measured to compare the efficiency of the two methods. Luciferase activity was measured according to the manufacturer's instructions (Luci ferase Assay Reagent, Pr omega), and the standard curve of the resultant was drawn, and then the number of intracellular exosomes was quantitatively calculated.

As shown in Figure 14, the method using the photospecific binding proteins CIBN and CRY2 of the present invention was confirmed that the effect of introducing into exosomes significantly higher than the conventional method XP (Fig. 14).

<5-2> Confirmation of target protein expression in produced axosome

After culturing the cells of Example <5-1> for 72 hours and separating the exosomes (Exoquick-TC, Systems biosciences), the exosomes isolated through the existing method XP and the method of the present invention As a result of indirectly comparing the amount of the target protein through the measurement of luciferase activity, as shown in FIG. 15, it was confirmed that the method of the present invention was able to produce a significant amount of the exosomeol containing the target protein significantly higher than the conventional method. (FIG. 15).

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Alternative Site (Article 26) <5-3> Comparison of introduction efficiency of target protein

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

[Equation 1]

_ |: Ί¾ Β¾ active wells of calculated exotransportation Luciferase m wells of exo transport production tax ¾

As shown in Figure 15, the exosomes produced using the combination of CRY2 and CIBN of the present invention was confirmed to be 4 to 120 times higher efficiency than the other comparative group (Fig. 15).

Example 6 Comparison of Axosome Delivery Efficiency to Target Cells

In order to compare the efficiency of the exosomes in which the target protein was introduced to the target cells, 5 x 10 ⁹ exosomes were treated in HeLa cells for 24 hours, and the fluorescence intensity expressed in the cells was measured. As shown, it was confirmed that the fluorescence intensity of the exosome (EXPLOR) of the present invention is significantly high (Fig. 16).

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

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Alternative Site (Article 26)

Claims

[Range of request]

[Claim 1]

a) introducing into a exosome-producing cell a polynucleotide encoding a fusion protein in a form in which an exosome-specific marker and a target protein are bound to obtain a transformed cell; And '

b) a method of producing a large amount of exosomes attached to the exosome membrane, the target protein, characterized in that it comprises culturing the transformed cells.

[Claim 2]

The method of claim 1, wherein the target protein is separated from the exosome membrane to produce a large amount of exosomes present in the exosomes.

[Claim 3] ^"

The method according to claim 1 or 2, wherein the exosome-producing cells are any one selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, and tumor cells. A method for producing a large amount of exosomes, characterized in that the above cells.

[Claim 4]

The method of claim 1, wherein the exosome specific marker is CD9, CD63, CD81 or CD82.

[Claim 5]

a) a polynucleotide encoding a fusion protein (a first fusion protein) in a form in which an exosome-specific marker and a first photospecific binding protein are bound to an exosome-producing cell and to bind with the first photospecific binding protein Introducing a polynucleotide encoding a fusion protein (second fusion protein) in a form in which the second photospecific binding protein which is capable of binding the desired protein;

b) irradiating the exosome-producing cells with light capable of causing the binding of the first photospecific binding protein to the second photospecific binding protein; And

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Alternative Site (Article 26) C) A method for producing a large amount of exosomes containing the target protein, comprising the step of stopping the irradiation of light after the exosomes are produced in the exosomes producing cells.

[Claim 6]

The method of claim 5, wherein the first photospecific binding protein is CIB (cryptochrome-interacting basichel ix ᅳ loop—he l.ix protein), CIBN (N-terminal domain of CIB), PhyB (hy t ochr ome B) Exosomes, characterized in that any one or more proteins selected from the group consisting of PIF (phytochrome interacting factor), FKFKFlavinbinding, Kelch repeat, Fbox 1), GIGANTEA, CRY (chryp to chrome) and PHRCphytolyase homolgous region How to manufacture in bulk.

[Claim 7]

The method of claim 5, wherein when the first photospecific binding protein is CIB or CIBN, the second photospecific binding protein is CRY or PHR, and the first photospecific binding protein and the second photospecific binding. The method of producing a large amount of exosomes, characterized in that the binding of the protein is performed by irradiation with light having a wavelength of 460 to 490 nm.

[Claim 8]

The method of claim 5, wherein when the first photospecific binding protein ¾ is PhyB, the second photospecific binding protein is PIF, and the first photospecific binding protein and the second photospecific binding protein are bound. Silver is a method for producing a large amount of exosomes, characterized in that carried out by irradiation with light having a wavelength of 600 to 650 nm.

[Claim 9]

The method of claim 5, wherein when the first photospecific binding protein is GIGANTEA, the second photospecific binding protein is F F1, wherein the crab 1 photospecific binding protein and the second photospecific binding protein Combining is performed by irradiating light having a wavelength of 460 to 490 nm in large quantities.

[Claim 10]

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Alternative Site (Article 26) A method for delivering a protein drug to the cytoplasm, characterized by using an exosome prepared by the method of claim 1, 2 or 5.

[Claim 11]

A pharmaceutical composition for cytoplasmic delivery of a protein drug comprising an exosome prepared by the method of claim 1, 2 or 5 as an active ingredient.

[Claim 12]

(a) a first expression vector comprising a polynucleotide encoding a fusion protein (first fusion protein) in a form in which an exosome specific marker and a first photospecific binding protein are bound;

(b) a second expression comprising a polyclonal site into which a polynucleotide encoding a protein of interest can be introduced and a polynucleotide encoding a second photospecific binding protein capable of binding to the first photospecific binding protein A vector for producing exosomes, including a vector.

[Claim 13]

The vector of claim 12, wherein the second expression vector expresses a fusion protein (second fusion protein) in a form in which the second photospecific binding protein and the target protein are fused in exosome producing cells. .

[Claim 14]

An exosome containing a target protein therein, prepared using the method of any one of claims 1, 2 or 5.

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Alternative Site (Article 26)