KR102100420B1 - Method for producing an exosome that transfers a substance specifically to target and exosome produced by the same method - Google Patents

Abstract

The present invention relates to a method for producing an exosome that delivers a target-specific substance and an exosome produced by the method. Specifically, an exosome prepared by introducing a vector in which a target peptide is inserted between the 170th and 171th amino acids of the CD9 protein of the present invention into an exosome-producing cell, the target peptide is expressed on the surface of the exosome, thereby exo By confirming that the drug trapped in the target is well delivered to the target tissue, the exosome can be usefully used as a target-specific transporter.

Description

METHOD FOR PRODUCING AN EXOSOME THAT TRANSFERS A SUBSTANCE SPECIFICALLY TO TARGET AND EXOSOME PRODUCED BY THE SAME METHOD}

The present invention relates to a method for producing an exosome that delivers a target-specific substance and an exosome produced by the method.

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

The cell is surrounded by a cell membrane of a double membrane structure composed of phospholipids, and the cell membrane prevents foreign matter from entering the cell. Most of the protein therapeutics developed so far do not enter the cytoplasm through the cell membrane, but act on the outside of the cell or act on the receptors on the cell membrane to transmit the signal to the cell, thereby exhibiting its activity.

Meanwhile, many proteins present in the cytoplasm interact with each other to regulate physiological activity. Therefore, if protein therapeutic agents can be directly injected into the cytoplasm, the activity of the cells can be more effectively controlled.

Recently, a method of directly introducing a target protein into cells has been studied. For example, a recombinant protein of a target protein and a protein transduction domain (PTD), which is a peptide that passes through the cell membrane, is made and the recombinant protein passes through the cell membrane and enters the cytoplasm. Cell membrane transduction domains include HIV-1 human immunodeficiency virus type-1 transacrivating regulatory protein (ATV), HSV VP22, Antp, dfTAT, and Hph-1. However, the above method does not properly refold the protein in the process of producing and separating the fusion protein in which the cell membrane transduction domain and the target protein are combined in the form of a recombinant protein, thus reducing the activity of the produced protein, and non-specific delivery , There is a problem that the yield is low.

On the other hand, target proteins combined with various nanoparticles such as gold nanoparticles (NP), liposome NPs, magnetic NPs, and polymeric NPs can enter the cytoplasm by passing through the cell membrane by endocytosis. However, the combination of the nanoparticle and the target protein is mostly degraded in the cell lysosome. When the target protein is decomposed inside the lysosome, the protein activity is lost. In addition, it is difficult to separate the target protein and the nanoparticles from the cytoplasm, and the toxicity of the nanoparticles can be a problem.

Exosomes (exosomes) refer to small vesicles with a membrane structure of 50-200 nm in size that are secreted out of cells while capturing proteins, DNA, RNA, etc., for intercellular signaling.

Exosomes were originally discovered in the process of leaving only hemoglobin in red blood cells by excreting and removing intracellular proteins in the final stages of red blood cell maturation. These exosomes are not directly separated from the plasma membrane in the study through electron microscopy, but originate from a specific compartment in a cell called a multivesicular body (MVB) and are released and secreted outside the cell. That is, when the fusion of the polycyst and the plasma membrane occurs, the vesicles are released into the extracellular environment, which is called exosome.

It is not clear what molecular mechanism these exosomes are produced by, but various types of immune cells and stem cells, including B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, etc. It is also known that tumor cells produce and secrete exosomes in a living state.

Exosomes include various proteins, DNA, and RNA in cells. As such, substances contained in the exosomes and secreted out of the cells are re-introduced into other cells by fusion or endocytosis with the cell membrane, and also serve as a communication between cells.

Exosomes containing a target protein therein can be used in the treatment of various diseases in vivo. For this, it is necessary to be able to efficiently produce exosomes containing the target protein. In Korean Patent Registration No. 10-0519384, a gene for a specific antigen is introduced into a cell line, and a protein produced from the introduced gene is stably expressed in the cell line, and then released into the cell through exosomes and the method described above A method of using the produced exosome as a vaccine is disclosed.

However, since the exosomes are naturally formed in cells, even if a gene encoding a target protein is introduced into cells generating exosomes, the likelihood that exosomes capturing the expressed protein is very low is very low. , There is a problem that the prepared exosomes are low in efficiency to be delivered to the target tissue.

CD9 is a cell surface glycoprotein receptor of about 24 to 27 kD belonging to the tetraspanin family, and modulates signaling functions important for regulating cell development, activity, growth and motility. It can also regulate cell adhesion and cell migration, and induce platelet activation involved in platelet-induced endothelial cell proliferation. In addition, it is involved in various phenomena in the cell, such as promoting muscle cell fusion and contributing to the maintenance of the root canal.

The tetraspanin family has four transmembrane sites, with both the N-terminal and C-terminal located in the intracellular region of the membrane, with two cells between the first and second, and third and fourth transmembrane sites. The extracellular loop is protruding.

Thus, the present inventors were developing a method for more effectively delivering exosomes to target tissues, and prepared a vector by inserting a target peptide between the 170th and 171th amino acids of the CD9 protein, which is mainly expressed in exosomes, and Introduced into exosome-producing cells to prepare exosomes with target peptides labeled on exosome membrane proteins. In addition, the present invention was completed by confirming that the peptide inserted into the exosome is well expressed in HEK293T cells, and that the drug captured in the exosome is well delivered to the target tissue.

An object of the present invention is to provide a method for preparing an exosome that delivers an active substance specifically for a target and an exosome prepared by the above method.

Another object of the present invention is to provide a method for delivering an active substance to a target tissue using exosomes prepared by the method of the present invention.

Another object of the present invention is to provide a pharmaceutical composition for the delivery of a protein drug comprising an exosome prepared by the method of the present invention as an active ingredient.

Another object of the present invention is to provide a composition for preparing an exosome comprising an expression vector in which a target peptide is inserted into the extracellular portion of tetraspanin.

In order to achieve the above object, the present invention comprises the steps of: 1) preparing an expression vector by inserting a target peptide into the extracellular portion of tetraspanin; And 2) introducing the expression vector of step 1) into exosome-producing cells.

In addition, the present invention provides an exosome that delivers an active substance specifically targeted by the method of the present invention.

In addition, the present invention provides a method of delivering an active substance to a target tissue using exosomes prepared by the method of the present invention.

In addition, the present invention provides a pharmaceutical composition for delivery of a protein drug comprising an exosome prepared by the method of the present invention as an active ingredient.

In addition, the present invention provides a composition for preparing an exosome comprising an expression vector in which a target peptide is inserted into the extracellular portion of tetraspanin.

The exosome prepared by introducing a vector into which the target peptide is inserted between the 170th and 171th amino acids of the CD9 protein of the present invention into an exosome-producing cell is a target peptide expressed on the surface of the exosome, thereby capturing the exosome By confirming that the drug is well delivered to the target tissue, the exosome can be usefully used as a target-specific delivery agent.

1 is a schematic diagram of a vector in which a gene encoding a protein in which a target peptide complex is inserted between the CIBN gene, the EGFP gene, and the 170th and 171th amino acids of the CD9 protein is inserted into the pSF-CMV-CMV-SbfI vector (A) , And FIG. (B) showing a site in which the target peptide complex is inserted in the CD9 protein structure.
Figure 2 is a photograph confirming the expression of the angiopeptin-2 peptide complex in HEK293T cells treated with an exosome labeled with an angiopeptin-2 peptide complex on a membrane protein.
FIG. 3 is a photograph confirming the expression of the apoB peptide complex in HEK293T cells treated with the exosome labeled with the apoB peptide complex on the membrane protein.
4 is a photograph confirming the expression of the apoE peptide complex in HEK293T cells treated with exosomes labeled with the apoE peptide complex membrane protein.
Figure 5 is a photograph confirming the expression of the VCAM-1 internalization sequence peptide complex in HEK293T cells treated with exosomes labeled with the VCAM-1 internalization sequence peptide complex membrane protein.
Figure 6 is a pSF-CMV-CMV-SbfI vector Cre recombinase-CRY2 gene, CIBN gene, EGFP gene, and a gene encoding a protein encoding a target peptide complex between the 170 and 171th amino acids of the CD9 protein is inserted It is a schematic diagram of a vector.
7 is a histopathologically confirming the target-specific delivery effect of the target peptide complex labeled exosomes on the membrane protein:
A: As a control, exosomes without a target peptide labeled on a membrane protein;
B: exosomes labeled with angiopeptin-2 peptide complex;
C: apoB peptide complex labeled exosomes;
D: ApoE peptide complex labeled exosomes.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of 1) preparing an expression vector by inserting a target peptide into an extracellular space of tetraspanin; And 2) introducing the expression vector of step 1) into exosome-producing cells.

As used herein, the term "tetraspanin (tetraspanin)" is a membrane protein that passes through the cell membrane 4 times, and may be a protein that exists on the cell membrane and exchanges information between cells and regulates cell proliferation. Specifically, the tetraspanine may be any one or more proteins selected from the group consisting of CD9, CD37, CD53, CD63, CD81 and CD82. In one embodiment of the present invention, the tetraspanin may be a CD9 protein.

As used herein, the term "target peptide" is a peptide capable of delivering a substance to a specific site in vivo, and is expressed on the surface of an exosome to allow the exosome to move to a specific tissue. That is, the target peptide according to the present invention can be used as long as it is a peptide known to move to a specific tissue. In one embodiment of the present invention, the target peptide may be an angiopeptin-2, apoB, apoE, VCAM-1 internalization sequence or a transverse muscle target. The target peptide may be inserted between the amino acids at positions 170 and 171 from the N-terminus of the CD9 protein having the amino acid sequence of SEQ ID NO: 8.

The expression vector is a recombinant vector capable of expressing a target peptide in a desired host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express a 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 the termination codon are generally considered to be part of a nucleotide sequence encoding a polypeptide, and when a gene construct is administered, an individual It must exhibit an action in and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

The term "operably linked (operably linked)" of the present invention refers to a state in which a nucleic acid sequence encoding a desired protein or RNA and a nucleic acid expression control sequence are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect the expression of the coding sequence. Operable linkages with expression vectors can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage can use enzymes and the like generally known in the art.

In addition, the expression vector may further include a selection marker. Selection markers are markers for the selection of transformed microorganisms or recombinant vectors, and can be used to confer selectable phenotypes such as drug resistance, nutritional demand, resistance to cytotoxic agents, or expression of surface proteins. When a vector containing a selectable marker is used, only the cells expressing the selectable marker survive in the environment in which the selective agent is treated, so that transformed cells can be selected. As a selection marker, an antibiotic resistance gene, specifically kanamycin, can be used, but any selection marker that can be used in the art can be used.

The exosome-producing cells may be any one or more selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, macronuclear cells, macrophages, stem cells, and tumor cells. In one embodiment of the present invention, the exosome producing cells may be HEK293T cells.

The term "active substance" as used herein refers to a substance that enhances or inhibits biological functions, and a substance capable of maintaining its balance when a substance that regulates the function of the human body is deficient or excessively secreted and exhibits abnormal conditions. it means.

In a specific embodiment of the present invention, the present inventors report that an angiopeptin-2, apoB, as a target peptide between the 170th and 171th amino acids of the CIBN gene, EGFP gene and CD9 protein in the pSF-CMV-CMV-SbfI vector, ApoE, VCAM-1 internalization sequence or transverse muscle target peptide was inserted to produce a vector, and the vector was introduced into HEK293T cells, which are exosome-producing cells, to obtain exosomes labeled with the target peptide on the membrane protein (FIG. 1), it was confirmed that the target peptide labeled on the membrane protein of the obtained exosome was well expressed (see FIGS. 2 to 5).

In addition, the present invention provides an exosome that delivers an active substance specifically targeted by the method of the present invention.

The method includes the steps of: 1) constructing an expression vector by inserting a target peptide into the extracellular portion of tetraspanin; And 2) introducing the expression vector of step 1) into exosome-producing cells.

The method may have the characteristics as described above. In one example, the tetraspanin may be any one or more proteins selected from the group consisting of CD9, CD37, CD53, CD63, CD81 and CD82. In one embodiment of the present invention, the tetraspanin may be a CD9 protein.

The target peptide can be any peptide that targets a specific tissue. In one embodiment of the present invention, the target peptide may be an angiopeptin-2, apoB, apoE, VCAM-1 internalization sequence or a transverse muscle target.

The exosome-producing cells may be any one or more selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, macronuclear cells, macrophages, stem cells, and tumor cells. In one embodiment of the present invention, the exosome producing cells may be HEK293T cells.

In a specific embodiment of the present invention, the present inventors construct a vector by inserting a target peptide complex between the 170th and 171th amino acids of the CIBN gene, EGFP gene, and CD9 protein in the pSF-CMV-CMV-SbfI vector, and The vector was introduced into HEK293T cells, which are exosome-producing cells, to obtain exosomes labeled with a target peptide on a membrane protein (see FIG. 1). It was confirmed that the target peptide labeled on the membrane protein of the obtained exosome was well expressed (see FIGS. 2 to 5).

In addition, the present invention provides a method of delivering an active substance to a target tissue using exosomes prepared by the method of the present invention.

The method includes the steps of: 1) constructing an expression vector by inserting a target peptide into the extracellular portion of tetraspanin; And 2) introducing the expression vector of step 1) into exosome-producing cells.

The method may have the characteristics as described above. In one example, the tetraspanin may be any one or more proteins selected from the group consisting of CD9, CD37, CD53, CD63, CD81 and CD82. In one embodiment of the present invention, the tetraspanin may be a CD9 protein.

The target peptide can be any peptide that targets a specific tissue. In one embodiment of the present invention, the target peptide may be an angiopeptin-2, apoB, apoE, VCAM-1 internalization sequence or a transverse muscle target.

The exosome-producing cells may be any one or more selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, macronuclear cells, macrophages, stem cells, and tumor cells. In one embodiment of the present invention, the exosome producing cells may be HEK293T cells.

In a specific embodiment of the present invention, the present inventors construct a vector by inserting a target peptide complex between the 170th and 171th amino acids of the CIBN gene, EGFP gene, and CD9 protein in the pSF-CMV-CMV-SbfI vector, and The vector was introduced into HEK293T cells, which are exosome-producing cells, to obtain exosomes labeled with a target peptide on a membrane protein (see FIG. 1). It was confirmed that the target peptide labeled on the membrane protein of the obtained exosome was well expressed (see FIGS. 2 to 5), and that the drug captured on the exosome was well delivered to the target tissue (see FIG. 7).

In addition, the present invention provides a pharmaceutical composition for delivery of a protein drug comprising an exosome prepared by the method of the present invention as an active ingredient.

The method includes the steps of: 1) constructing an expression vector by inserting a target peptide into the extracellular portion of tetraspanin; And 2) introducing the expression vector of step 1) into exosome-producing cells.

The method may have the characteristics as described above. In one example, the tetraspanin may be any one or more proteins selected from the group consisting of CD9, CD37, CD53, CD63, CD81 and CD82. In one embodiment of the present invention, the tetraspanin may be a CD9 protein.

The target peptide can be any peptide that targets a specific tissue. In one embodiment of the present invention, the target peptide may be an angiopeptin-2, apoB, apoE, VCAM-1 internalization sequence or a transverse muscle target.

The exosome-producing cells may be any one or more selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, macronuclear cells, macrophages, stem cells, and tumor cells. In one embodiment of the present invention, the exosome producing cells may be HEK293T cells.

In a specific embodiment of the present invention, the present inventors construct a vector by inserting a target peptide complex between the 170th and 171th amino acids of the CIBN gene, EGFP gene, and CD9 protein in the pSF-CMV-CMV-SbfI vector, and The vector was introduced into HEK293T cells, which are exosome-producing cells, to obtain exosomes labeled with a target peptide on a membrane protein (see FIG. 1). It was confirmed that the target peptide labeled on the membrane protein of the obtained exosome was well expressed (see FIGS. 2 to 5), and that the drug captured on the exosome was well delivered to the target tissue (see FIG. 7).

The pharmaceutical composition may include 10 to 95% by weight of exosomes as an active ingredient relative to the total weight of the pharmaceutical composition. In addition, the pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function in addition to the active ingredients.

The compositions of the present invention may also include carriers, diluents, excipients, or combinations of two or more of those commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or the like described above may be mixed with one or more of these components. At this time, other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.

When formulating the composition, it is usually prepared using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants.

The composition of the present invention may be formulated as oral preparations or parenteral preparations. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations may contain at least one excipient in one or more compositions, such as starch, calcium carbonate, sucrose, It can be prepared by mixing lactose and gelatin. Also, lubricants such as magnesium stearate and talc may be added. On the other hand, the liquid formulation includes a suspension, an intravenous solution, an emulsion or a syrup, etc., which may include excipients such as wetting agents, sweeteners, fragrances, preservatives, and the like.

Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, injections such as emulsions.

As non-aqueous solvents and suspension solvents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally depending on the desired method, and parenteral administration is either for external use for skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection. Can be selected.

The composition according to the invention is administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, drug activity, sensitivity to the drug, administration time, administration route and discharge rate, treatment period, and drugs used simultaneously. The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration may be sequential or simultaneous.

However, for the desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.001 to 10,000 mg / kg, specifically 0.1 to 5 g / kg. The administration may be once a day or divided into several times.

In addition, the present invention provides a composition for preparing an exosome comprising an expression vector in which a target peptide is inserted into the extracellular portion of tetraspanin.

The tetraspanine may be any one or more proteins selected from the group consisting of CD9, CD37, CD53, CD63, CD81 and CD82. In one embodiment of the present invention, the tetraspanin may be a CD9 protein.

The target peptide can be any peptide that targets a specific tissue. In one embodiment of the present invention, the target peptide may be an angiopeptin-2, apoB, apoE, VCAM-1 internalization sequence or a transverse muscle target.

The expression vector may have the characteristics as described above. In one example, the expression vector is a recombinant vector capable of expressing a target peptide in a desired host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express a gene insert. In addition, the expression vector may further include a selection marker. As a selection marker, an antibiotic resistance gene, specifically kanamycin, can be used, but any selection marker that can be used in the art can be used.

The composition is one or more other component compositions, solutions or devices suitable for introduction of expression vectors, culturing transformed cells for producing exosomes, and isolating and purifying exosomes produced from transformed cells for producing exosomes May be included. For example, an appropriate buffer required for introduction of the expression vector, a medium and a container required for culturing the transformed cells for producing exosomes, and the like may be further included.

In a specific embodiment of the present invention, the present inventors construct a vector by inserting a target peptide complex between the 170th and 171th amino acids of the CIBN gene, EGFP gene, and CD9 protein in the pSF-CMV-CMV-SbfI vector, and The vector was introduced into HEK293T cells, which are exosome-producing cells, to obtain exosomes labeled with a target peptide on a membrane protein (see FIG. 1). It was confirmed that the target peptide labeled on the membrane protein of the obtained exosome was well expressed (see FIGS. 2 to 5), and that the drug captured on the exosome was well delivered to the target tissue (see FIG. 7).

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only to illustrate the present invention, and the contents of the present invention are not limited by them.

Preparation of exosomes labeled with an angiopeptin-2 peptide complex on a membrane protein

Angiopeptin-2 is a protein targeting the blood-brain barrier, and an exosome in which angiopeptin-2 peptide is labeled on a membrane protein was prepared in the following manner.

First, the DNA was linearized by cutting the NdeI portion of the multicloning site of the pSF-CMV-CMV-SbfI (# OG411, Oxford Genetics, UK) vector using an NdeI restriction enzyme. Subsequently, a fragment in which the genes encoding the 1st to 170th amino acids from the N-terminal of the CDB protein and the EGFP gene described as the nucleotide sequence of SEQ ID NO: 10 and the nucleotide sequence of SEQ ID NO: 10 are sequentially linked through PCR. , Gene fragments encoding the 171 th to 228 th amino acids from the N-terminus of the CD9 protein, and gene fragments encoding the angiopeptin-2 peptide complex were constructed. Next, the NdeI portion of the pSF-CMV-CMV-SbfI vector was sequentially connected by the Gibson assembly method so that both ends of the three fragments overlapped with each other by 20 to 24 bp, CIBN gene, EGFP CIBN-EGFP-CD9 (1-170) -anne, in which a gene encoding the angiopeptin-2 peptide complex described by the amino acid sequence of SEQ ID NO: 1 was inserted between the 170th and 171st amino acids of the gene and the CD9 protein Geopeptin 2 complex-CD9 (171-228) vector was constructed (FIG. 1). In the angiopeptin-2 peptide complex, the amino acid sequence of angiopeptin-2 is repeated three times, and between each angiopeptin-2 amino acid sequence is a linker described as an amino acid sequence of GGGGS, and an angiopeptin-2 amino acid sequence. At both ends, a linker described by the amino acid sequence of PPVAT is inserted. Thereafter, CIBN-EGFP-CD9 (1-170) -Angiopeptin 2 complex-CD9 (171-228) vector was introduced into HEK293T cells, which are exosome-producing cells, and cultured for 24 hours, followed by fetal bovine serum. The culture was further incubated for 48 hours by changing to a medium not included. After the culture was collected by centrifugation at 1,000 rpm for 3 minutes, the culture was filtered using a polyethersulfone membrane having a pore size of 0.2 μm. The filtrate was first concentrated through tangential flow filtration at 4 ° C. Then, the concentrate was purified using size exclusion chromatography consisting of sepharose beads at 4 ° C. Next, after adding and diluting 300 to 500 ml of a 4 ° C phosphate buttered saline aqueous solution in a purification solution, performing secondary concentration through tangential flow filtration at 4 ° C, and Geopeptin-2 peptide complexes obtained exosomes labeled with a membrane protein.

Preparation of exosomes labeled with apoB (apoB) peptide complex on membrane protein

ApoB is a protein targeting the blood-brain barrier, and the apoB peptide complex was prepared in the following manner by exosomes labeled on the membrane protein.

The amino acid sequence of ApoB is repeated three times, and between each ApoB amino acid sequence, a linker described as an amino acid sequence of GGGGS is inserted, and at both ends, a linker described as an amino acid sequence of PPVAT is inserted at both ends. Exosome labeled with the membrane protein was obtained in the same manner as in <Example 1>, except that the ApoB peptide complex described by the amino acid sequence was used.

Preparation of apoE (apoE) peptide complex labeled exosomes on membrane protein

ApoE is a protein targeting the blood-brain barrier, and the apoE peptide complex was prepared by the following method for exosomes labeled on the membrane protein.

The amino acid sequence of ApoE is repeated three times, and between each ApoE amino acid sequence, a linker described by the amino acid sequence of GGGGS is inserted, and a linker described by the amino acid sequence of PPVAT is inserted at both ends of the ApoE amino acid sequence. Exosome labeled with the membrane protein was obtained in the same manner as in <Example 1>, except that the apoE peptide complex described by the amino acid sequence was used.

Preparation of exosomes labeled with VCAM-1 internalization sequence peptide complex on membrane protein

VCAM-1 (vascular cell adhesion molecule-1) is a protein targeting a site of vascular inflammation, and the VCAM-1 internalization sequence peptide was prepared in the following manner as a membrane protein-labeled exosome.

VCAM-1 internalization sequence is repeated three times, between each VCAM-1 internalization sequence is a linker described by the amino acid sequence of GGGGS, and a linker described by the amino acid sequence of PPVAT is inserted at both ends of the VCAM-1 internalization sequence. Except that the VCAM-1 internalization sequence peptide complex described by the amino acid sequence of 4 was used, the exosome labeled with the VCAM-1 internalization sequence peptide complex was membrane protein was obtained in the same manner as in <Example 1>.

Preparation of exosomes labeled with transverse muscle target peptide complex on membrane protein

The striated muscle target peptide is a peptide that targets the striated muscle site, and the exosomes labeled with the striated muscle target peptide complexes are prepared in the following manner.

In the transverse muscle target peptide complex, the ASSLNIA, TARGEHKEEELI or SKTFNTHPQSTP amino acid sequence is repeated three times, and between each ASSLNIA, TARGEHKEEELI or SKTFNTHPQSTP amino acid sequence is a linker described by the amino acid sequence of GGGGS, and the ASSLNIA, TARGEHKEEELI or SKTFNTHPQSTP amino acid sequence at the end of the ASSLNIA, TARGEHKEEELI or SKTFNTHPQSTP amino acid sequence Transverse in the same manner as in <Example 1>, except that the transverse muscle target peptide complex described by the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 into which the linker described by the amino acid sequence of PPVAT is inserted is inserted. Patterned muscle target peptide complex to obtain an exosome labeled on the membrane protein.

<Experimental Example 1> Confirmation of expression of angiopeptin-2 peptide complex

After the exosome of <Example 1> was treated to HEK293T cells, expression of angiopeptin-2 peptide complex labeled on the membrane protein of exosome was confirmed through a fluorescent microscope 24 hours later.

As a result, as shown in FIG. 2, it was confirmed that the angiopeptin-2 peptide complex was expressed on the surface of the exosome (FIG. 2).

<Experimental Example 2> Confirmation of the expression of the ApoB peptide complex

After the exosome of <Example 2> was treated to HEK293T cells, expression of the apoB peptide complex labeled on the membrane protein of the exosome was confirmed through a fluorescent microscope after 24 hours.

As a result, as shown in Figure 3, it was confirmed that the ApoB peptide complex is expressed on the surface of the exosome (Figure 3).

<Experiment 3> Confirmation of the expression of the apoE peptide complex

After the exosome of <Example 3> was treated to HEK293T cells, expression of the apoE peptide complex labeled on the exosome membrane protein was confirmed through a fluorescent microscope after 24 hours.

As a result, as shown in Figure 4, it was confirmed that the apoE peptide complex is expressed on the surface of the exosome (Figure 4).

<Experimental Example 4> VCAM-1 internalization sequence confirmed the expression of the peptide complex

After treating the exosome of <Example 4> with HEK293T cells, expression of the VCAM-1 internalization sequence peptide complex labeled on the exosome membrane protein was confirmed through a fluorescence microscope after 24 hours.

As a result, as shown in Figure 5, it was confirmed that the VCAM-1 internalization sequence peptide complex is expressed on the surface of the exosome (Figure 5).

<Experimental Example 5> Confirmation of the expression of the transverse muscle target peptide complex

After the exosome of <Example 5> was treated to HEK293T cells, expression of the transverse muscle target peptide complex labeled on the exosome membrane protein was confirmed through a fluorescent microscope after 24 hours.

As a result, it was confirmed that the transverse muscle target peptide complex was expressed on the surface of the exosome.

<Experimental Example 6> Target specific delivery effect of an exosome labeled with an angiopeptin-2 peptide complex on a membrane protein

CIBN-EGFP-CD9 (1) manufactured in the same manner as in <Example 1>, except that a Cre recombinase-CRY2 gene was additionally inserted under an LED light emitting a light of 460 nm with an intensity of 100 μW. -170) -Angiopeptin 2 complex-CD9 (171-228) vector (Fig. 6) was introduced into HEK293T cells, exosome producing cells. Cre recombinase was used as the active substance to be delivered. After culturing the cells for 24 hours, the medium was replaced with fetal calf-free medium, and further cultured for 48 hours under the LED. Thereafter, the culture medium was separated, and tangential flow filtration and size exclusion chromatography were performed to obtain an exosome labeled with an angiopeptin-2 peptide complex on the membrane protein. At this time, as a control, HEK293T cells obtained an exosome not labeled with a membrane protein angiopeptin-2 peptide complex and used. The obtained 1 × 10 ⁹ particles / 50 μl concentration of exosomes was veined into a blood vessel of a Cre reporter mouse C57BL / 6 loxP-eNphr3.0-loxP-eYFP TG mouse (The Jackson Laboratory, Bar Harbor, Maine, USA) or 48-72 hours after intraperitoneal injection, the organs of the mouse were removed for histopathological examination, and the eYFP distribution of each mouse was analyzed to analyze the distribution of exosomes in which specific target peptides are labeled on the membrane protein in vivo. And functionality.

As a result, as shown in FIG. 7, it was confirmed that the exosome labeled with the angiopeptin-2 peptide complex on the membrane protein is specifically well delivered to the blood-brain barrier (FIG. 7B).

<Experimental Example 7> Target specific delivery effect of exosomes labeled with apoB peptide complex on membrane protein

CIBN-EGFP-CD9 (1) manufactured in the same manner as in <Example 2>, except that a Cre recombinase-CRY2 gene was further inserted under an LED light emitting a light of 460 nm with an intensity of 100 μW. -170) -ApoB peptide complex-In the same manner as in <Experiment 6>, except that the CD9 (171-228) vector (FIG. 6) was used, in vivo of exosomes in which a specific target peptide was labeled on a membrane protein Delivery distribution and function were verified.

As a result, as shown in FIG. 7, it was confirmed that the exosomes labeled with the apoB peptide complex on the membrane protein are specifically well delivered to the blood-brain barrier (FIG. 7C).

<Experimental Example 8> Target specific delivery effect of exosomes labeled with apoE peptide complex on membrane protein

CIBN-EGFP-CD9 (1) manufactured in the same manner as in <Example 3> except that an additional Cre recombinase-CRY2 gene was additionally inserted under an LED light emitting a light of 460 nm with an intensity of 100 μW. -170) -ApoE peptide complex-CD9 (171-228) In the same manner as in <Experiment 6>, except that the vector (FIG. 6), a specific target peptide in vivo of the exosomes labeled on the membrane protein Delivery distribution and function were verified.

As a result, as shown in FIG. 7, it was confirmed that the exosomes labeled with the apoE peptide complex on the membrane protein are specifically well delivered to the blood-brain barrier (D in FIG. 7).

<Experimental Example 9> Target specific delivery effect of exosomes labeled with a VCAM-1 internalization sequence peptide complex on membrane proteins

CIBN-EGFP-CD9 (1) manufactured in the same manner as in <Example 4> except that an additional Cre recombinase-CRY2 gene was further inserted under an LED light emitting a light of 460 nm with an intensity of 100 μW. -170) -VCAM 1 internalization sequence Peptide complex-CD9 (171-228) In the same manner as in <Experiment 6> except that the vector (Fig. 6), the specific target peptide of the exosomes labeled on the membrane protein In vivo delivery distribution and function were verified.

As a result, it was confirmed that the exosomes labeled with the VCAM-1 internalization sequence peptide complex on the membrane protein are specifically well delivered to the site of vascular inflammation.

<Experimental Example 10> Target specific delivery effect of exosomes labeled with a transverse muscle target peptide complex on a membrane protein

CIBN-EGFP-CD9 (1) manufactured in the same manner as in <Example 5>, except that a Cre recombinase-CRY2 gene was additionally inserted under an LED light emitting a light of 460 nm with an intensity of 100 μW. -170) -Horizontal muscle target peptide complex-In the same manner as in <Experiment 6>, except that the CD9 (171-228) vector (FIG. 6) was used, the specific target peptide of the exosome labeled with the membrane protein In vivo delivery distribution and function were verified.

As a result, it was confirmed that the exosomes labeled with the transverse muscle target peptide complex on the membrane protein were specifically well delivered to the transverse muscle site.

<110> Korea Advanced Institute of Science and Technology          Cellex Life Sciences, Incorporated <120> METHOD FOR PRODUCING AN EXOSOME THAT TRANSFERS A SUBSTANCE          SPECIFICALLY TO TARGET AND EXOSOME PRODUCED BY THE SAME METHOD <130> 2018P-08-010 <150> KR 10-2017-0104171 <151> 2017-08-17 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> angiopeptin 2 peptide complex <400> 1 Pro Pro Val Ala Thr Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg   1 5 10 15 Asn Asn Phe Lys Thr Glu Glu Tyr Gly Gly Gly Gly Ser Thr Phe Phe              20 25 30 Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr Glu Glu Tyr          35 40 45 Gly Gly Gly Gly Ser Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg      50 55 60 Asn Asn Phe Lys Thr Glu Glu Tyr Pro Pro Val Ala Thr  65 70 75 <210> 2 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> ApoB peptide complex <400> 2 Pro Pro Val Ala Thr Ser Ser Val Ile Asp Ala Leu Gln Tyr Lys Leu   1 5 10 15 Glu Gly Thr Thr Arg Leu Thr Arg Lys Arg Gly Leu Lys Leu Ala Thr              20 25 30 Ala Leu Ser Leu Ser Asn Lys Phe Val Glu Gly Ser Gly Gly Gly Gly          35 40 45 Ser Ser Ser Val Ile Asp Ala Leu Gln Tyr Lys Leu Glu Gly Thr Thr      50 55 60 Arg Leu Thr Arg Lys Arg Gly Leu Lys Leu Ala Thr Ala Leu Ser Leu  65 70 75 80 Ser Asn Lys Phe Val Glu Gly Ser Gly Gly Gly Gly Ser Ser Ser Val                  85 90 95 Ile Asp Ala Leu Gln Tyr Lys Leu Glu Gly Thr Thr Arg Leu Thr Arg             100 105 110 Lys Arg Gly Leu Lys Leu Ala Thr Ala Leu Ser Leu Ser Asn Lys Phe         115 120 125 Val Glu Gly Ser Pro Pro Val Ala Thr     130 135 <210> 3 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> ApoE peptide complex <400> 3 Pro Pro Val Ala Thr Leu Arg Lys Leu Arg Lys Arg Leu Leu Leu Arg   1 5 10 15 Lys Leu Arg Lys Arg Leu Leu Gly Gly Gly Gly Ser Leu Arg Lys Leu              20 25 30 Arg Lys Arg Leu Leu Leu Arg Lys Leu Arg Lys Arg Leu Leu Gly Gly          35 40 45 Gly Gly Ser Leu Arg Lys Leu Arg Lys Arg Leu Leu Leu Arg Lys Leu      50 55 60 Arg Lys Arg Leu Leu Pro Pro Val Ala Thr  65 70 <210> 4 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> VCAM 1 internalizing sequence peptide complex <400> 4 Pro Pro Val Ala Thr Val His Pro Lys Gln His Arg Gly Gly Gly Gly   1 5 10 15 Ser Val His Pro Lys Gln His Arg Gly Gly Gly Gly Ser Val His Pro              20 25 30 Lys Gln His Arg Pro Pro Val Ala Thr          35 40 <210> 5 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> striated muscle targeting peptide complex <400> 5 Pro Pro Val Ala Thr Ala Ser Ser Leu Asn Ile Ala Gly Gly Gly Gly   1 5 10 15 Ser Ala Ser Ser Leu Asn Ile Ala Gly Gly Gly Gly Ser Ala Ser Ser              20 25 30 Leu Asn Ile Ala Pro Pro Val Ala Thr          35 40 <210> 6 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> striated muscle targeting peptide complex <400> 6 Pro Pro Val Ala Thr Thr Ala Arg Gly Glu His Lys Glu Glu Glu Leu   1 5 10 15 Ile Gly Gly Gly Gly Ser Thr Ala Arg Gly Glu His Lys Glu Glu Glu              20 25 30 Leu Ile Gly Gly Gly Gly Ser Thr Ala Arg Gly Glu His Lys Glu Glu          35 40 45 Glu Leu Ile Pro Pro Val Ala Thr      50 55 <210> 7 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> striated muscle targeting peptide complex <400> 7 Pro Pro Val Ala Thr Ser Lys Thr Phe Asn Thr His Pro Gln Ser Thr   1 5 10 15 Pro Gly Gly Gly Gly Ser Ser Lys Thr Phe Asn Thr His Pro Gln Ser              20 25 30 Thr Pro Gly Gly Gly Gly Ser Ser Lys Thr Phe Asn Thr His Pro Gln          35 40 45 Ser Thr Pro Pro Pro Val Ala Thr      50 55 <210> 8 <211> 228 <212> PRT <213> Homo sapiens <400> 8 Met Pro Val Lys Gly Gly Thr Lys Cys Ile Lys Tyr Leu Leu Phe Gly   1 5 10 15 Phe Asn Phe Ile Phe Trp Leu Ala Gly Ile Ala Val Leu Ala Ile Gly              20 25 30 Leu Trp Leu Arg Phe Asp Ser Gln Thr Lys Ser Ile Phe Glu Gln Glu          35 40 45 Thr Asn Asn Asn Asn Ser Ser Phe Tyr Thr Gly Val Tyr Ile Leu Ile      50 55 60 Gly Ala Gly Ala Leu Met Met Leu Val Gly Phe Leu Gly Cys Cys Gly  65 70 75 80 Ala Val Gln Glu Ser Gln Cys Met Leu Gly Leu Phe Phe Gly Phe Leu                  85 90 95 Leu Val Ile Phe Ala Ile Glu Ile Ala Ala Ala Ile Trp Gly Tyr Ser             100 105 110 His Lys Asp Glu Val Ile Lys Glu Val Gln Glu Phe Tyr Lys Asp Thr         115 120 125 Tyr Asn Lys Leu Lys Thr Lys Asp Glu Pro Gln Arg Glu Thr Leu Lys     130 135 140 Ala Ile His Tyr Ala Leu Asn Cys Cys Gly Leu Ala Gly Gly Val Glu 145 150 155 160 Gln Phe Ile Ser Asp Ile Cys Pro Lys Lys Asp Val Leu Glu Thr Phe                 165 170 175 Thr Val Lys Ser Cys Pro Asp Ala Ile Lys Glu Val Phe Asp Asn Lys             180 185 190 Phe His Ile Ile Gly Ala Val Gly Ile Gly Ile Ala Val Val Met Ile         195 200 205 Phe Gly Met Ile Phe Ser Met Ile Leu Cys Cys Ala Ile Arg Arg Asn     210 215 220 Arg Glu Met Val 225 <210> 9 <211> 528 <212> DNA <213> Artificial Sequence <220> <223> CIBN gene <400> 9 atgaatggag ctataggagg tgaccttttg ctcaattttc ctgacatgtc ggtcctagag 60 cgccaaaggg ctcacctcaa gtacctcaat cccacctttg attctcctct cgccggcttc 120 tttgccgatt cttcaatgat taccggcggc gagatggaca gctatctttc gactgccggt 180 ttgaatcttc cgatgatgta cggtgagacg acggtggaag gtgattcaag actctcaatt 240 tcgccggaaa cgacgcttgg gactggaaat ttcaaggcag cgaagtttga tacagagact 300 aaggattgta atgaggcggc gaagaagatg acgatgaaca gagatgacct agtagaagaa 360 ggagaagaag agaagtcgaa aataacagag caaaacaatg ggagcacaaa aagcatcaag 420 aagatgaaac acaaagccaa gaaagaagag aacaatttct ctaatgattc atctaaagtg 480 acgaaggaat tggagaaaac ggattatatt catgtaccgg tcgccacc 528 <210> 10 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> EGFP gene <400> 10 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 720 agtggttccg gactcagatc tcgagctcaa gcttcgaatt ctgcagtcga cggtaccgcg 780 ggcccgggat ccaccggatc tagatca 807

Claims (13)

1) By inserting a nucleic acid encoding a target peptide into a nucleic acid encoding an extracellular space of tetraspanin, a fusion peptide having a target peptide bound to an extracellular portion of tetraspanin can be expressed. Constructing an expression vector;
2) introducing the expression vector of step 1) into exosome producing cells; And
3) culturing the exosome-producing cells to express the fusion peptide, wherein the target peptide is expressed on the surface of the exosome, a method for producing an exosome that specifically delivers an active substance to a target. .
The method of claim 1, wherein the tetraspanin is any one or more selected from the group consisting of CD9, CD37, CD53, CD63, CD81, and CD82, and a method for producing an exosome that specifically delivers an active substance.
The method of claim 2, wherein a target peptide is inserted between the amino acids located at positions 170 and 171 from the N-terminus of the CD9 protein.
The method of claim 2, wherein the CD9 protein has the amino acid sequence of SEQ ID NO: 8, and specifically, a method for producing an exosome that delivers a target-specific active substance.
According to claim 1, The exosome-producing cells are any one or more cells selected from the group consisting of B-lymphocytes, T-lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, and tumor cells. Method for producing an exosome that delivers an active substance specifically for a target.
According to claim 1, The target peptide is a target-specific active material characterized in that it is selected from the group consisting of angiopeptin-2, apoB, apoE, VCAM-1 internalization sequences and transverse muscle target peptide complexes. Method for manufacturing exosomes to be delivered.
An exosome that delivers a target-specifically active substance prepared by the method of any one of claims 1 to 6.
A pharmaceutical composition for the delivery of a protein drug comprising an exosome prepared as a method according to any one of claims 1 to 6 as an active ingredient.
An exosome in which a fusion peptide having a target peptide bound to an extracellular portion of tetraspanin is expressed.
The exosome according to claim 9, wherein the tetraspanin is at least one selected from the group consisting of CD9, CD37, CD53, CD63, CD81 and CD82.
The exosome according to claim 10, wherein a target peptide is inserted between the amino acids at positions 170 and 171 from the N-terminus of the CD9 protein.
The exosome according to claim 10, wherein the CD9 protein has the amino acid sequence of SEQ ID NO: 8.
The exosome according to claim 9, wherein the target peptide is selected from the group consisting of angiopeptin-2, apoB, apoE, VCAM-1 internalization sequence and transverse muscle target peptide complex.

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