Egosomes containing photocleavable proteins and their uses

Abstract

This invention relates to ectosomes containing photocleavable proteins and their uses. The ectosomes according to the invention comprise a fusion protein containing blue fluorescent protein (TagBFP), a photocleavable protein (mMaple3), and an ectosome-specific marker protein (CD9). It has been determined that when the ectosome is illuminated with 405 nm light, mMaple3, as the photocleavable protein, is cleaved, allowing the blue fluorescent protein in the ectosome to be delivered to target cells. Furthermore, it has been determined that when 405 nm light is applied to the ectosome containing the Cre fusion protein (Cre-mMaple3-CD9), the Cre protein in the ectosome can be delivered to animal organs. Therefore, it is anticipated that the ectosomes containing photocleavable proteins according to the invention will safely and efficiently deliver a variety of therapeutic proteins into cells, and thus be effectively used in the field of protein therapy.

Description

Technical Field

[0001] This disclosure relates to ectosomes containing photocleavable proteins and their uses, and more specifically, to ectosomes containing fusion proteins comprising a target protein and a photocleavable protein (mMaple3) and their uses.

[0002] This application claims priority to Korean Patent Application No. 10-2020-0015696, filed on February 10, 2020, and Korean Patent Application No. 10-2021-0015837, filed on February 4, 2021, and the entire contents of the description and drawings of the respective applications are incorporated herein by reference. Background Technology

[0003] Most current protein therapies target cell membrane proteins. However, the pathogens of many diseases reside primarily inside cells, and technologies for delivering therapeutic proteins into cells are needed to target these pathogens.

[0004] Therefore, many methods for directly introducing target proteins into cells have been investigated recently. One of these, the technique of delivering therapeutic proteins using lipid nanoparticles, has the problem that lipid nanoparticles cannot be effectively separated from therapeutic proteins. Another technique uses protein transduction domains (PTDs) that play a major role in the intracellular penetration of viruses. The problem is that PTDs are degraded when exposed to body fluids (e.g., blood or intestinal fluid).

[0005] Therefore, there is a need for technologies to efficiently deliver therapeutic proteins into cells in order to target pathogenic factors of specific diseases present inside cells.

[0006] On the other hand, efflux bodies are vesicles composed of a lipid bilayer and are components of substances secreted by cells to the extracellular space. Efflux bodies are known to play functional roles in mediating cell-cell communication and cellular immunity by transporting (delivering) proteins, bioactive lipids, and RNA (miRNA) (all of which are cellular biomolecules). These efflux bodies are also being studied as biomarkers for neurological diseases (such as Alzheimer's disease) and are being used to develop drug delivery systems, such as nanocarriers for specific drugs, due to their high selectivity in penetrating the blood-brain barrier (BBB) ​​that separates cerebrospinal fluid from blood.

[0007] Regarding techniques for delivering therapeutic proteins, Korean Patent Publication No. 10-2018-0036134 discloses a method for preparing ectosomes containing the hyperrepressor Iκ protein using a light-specific binding protein, and a pharmaceutical composition for the prevention and treatment of inflammatory diseases, comprising the ectosome prepared by the preparation method as an active ingredient; and Japanese Patent Application Publication No. 2019-528674 discloses a method for mass-producing ectosomes containing carrier proteins, a carrier for preparing ectosomes, ectosomes containing carrier proteins prepared by the method, and a method for loading carrier proteins into a cytosol using the ectosomes thus prepared.

[0008] However, the aforementioned documents disclose light-specific binding proteins as components of the fusion protein in the ectosome, and this uses two light-specific binding proteins, such as the CIBN-CRY2 system. Therefore, when the two constructs are expressed in cells, they must be co-transfected, thus reducing efficiency. Furthermore, continuous application of 488 nm light to the cells is required during ectosome generation, which can affect the cells and result in a loss of the amount of carrier protein contained in the ectosome. Additionally, there are the following drawbacks: once the ectosome is formed, the carrier protein cannot move freely when maintaining the binding of CIBN and CRY2 in the absence of light, and can bind to ectosome-specific proteins; the CRY2 fused to the carrier protein is as large as 65 kDa, thus affecting the inherent function of the carrier protein; and to determine whether it is well expressed in cells, it must be fused separately and a fluorescent protein (e.g., EGFP) must be used, thus making it impossible to determine whether the binding of CRY2 and CIBN is detached in the absence of light.

[0009] Therefore, the inventors sought to overcome the shortcomings of conventional known protein delivery technologies using the CIBN-CRY2 system and to develop a method for safely and efficiently delivering proteins into cells. Summary of the Invention

[0010] Technical issues

[0011] The inventors discovered that when an efflux body containing a combination of a photocleavable protein mMaple3 and a target protein to be delivered to a cell is irradiated, mMaple3 is cleaved to safely and efficiently release the target protein from the efflux body into the target cell, thereby completing the present disclosure.

[0012] Therefore, one object of this disclosure is to provide ectosomes comprising fusion proteins containing target proteins, photocleavable proteins and ectosome-specific marker proteins, and their uses.

[0013] However, the technical problems addressed by this disclosure are not limited to those described above, and other unmentioned problems will be clearly understood by those skilled in the art from the following description.

[0014] Technical solution

[0015] To achieve the above objectives, this disclosure provides an efflux body comprising a fusion protein containing a target protein and mMaple3.

[0016] Additionally, this disclosure provides compositions for delivering target proteins into cells, comprising efflux bodies as active ingredients.

[0017] Furthermore, this disclosure provides a method for delivering a target protein into cells in vitro, comprising irradiating an efflux body containing a fusion protein comprising the target protein and mMaple3 with light; and

[0018] Target cells were treated with efflux bodies that had been irradiated by light.

[0019] In addition, this disclosure provides a method for screening protein drug candidates for delivery to cells, comprising (a) irradiating an efflux body containing a fusion protein comprising a protein drug candidate and mMaple3 with light;

[0020] (b) Treatment of target cells with light-irradiated ectosomes; and

[0021] (c) When mMaple3 exhibits fluorescence in the target cells, it is determined that the protein candidate drug has been delivered to the cells.

[0022] As one embodiment of this disclosure, the fusion protein may also include ectosome-specific marker proteins.

[0023] In addition, this disclosure provides a method for preparing fusion proteins, which includes the following steps:

[0024] (S1) Amplify the cDNA of the target protein and mMaple3 respectively;

[0025] (S2) The amplified cDNAs of the target protein and mMaple3 are combined into a single cDNA to prepare a cDNA encoding a fusion protein containing the target protein and mMaple3; and

[0026] (S3) The fusion protein is expressed by introducing the cDNA encoding the fusion protein into a vector and then transfecting it into cells.

[0027] Furthermore, this disclosure provides a method for preparing efflux bodies, comprising the following steps:

[0028] (S1) Amplify the cDNA of the target protein and mMaple3 respectively;

[0029] (S2) The amplified cDNAs of the target protein and mMaple3 are combined into a single cDNA to prepare a cDNA encoding a fusion protein containing the target protein and mMaple3; and

[0030] (S3) The cDNA encoding the fusion protein was transfected into effusor-producing cells, and the effusors were isolated and purified from the cell culture medium.

[0031] As one embodiment of this disclosure, it may include combining the amplified cDNA of mMaple3 and the cDNA of the ectosome-specific marker protein into a single cDNA before combining the target protein and the amplified cDNA of mMaple3 into a single cDNA in (S2).

[0032] As another embodiment of this disclosure, the separation of ectosomes in (S3) may be performed using a method selected from TFF (tangential flow filtration), ultracentrifugation, size exclusion chromatography, and ectosome separation kits.

[0033] As another embodiment of this disclosure, mMaple3 may contain the amino acid sequence of SEQ ID NO:1.

[0034] As another embodiment of this disclosure, mMaple3 may be encoded by a gene containing the nucleotide sequence of SEQ ID NO:2.

[0035] As another embodiment of this disclosure, the target protein may be a protein to be delivered into the cell.

[0036] As another embodiment of this disclosure, the target protein may be a protein used to treat a disease or a protein used to diagnose a disease.

[0037] As another embodiment of this disclosure, the efflux-specific marker protein may be one or more selected from CD9, CD63 and CD81.

[0038] As another embodiment of this disclosure, the mMaple3 in the efflux body can be cut by irradiating the efflux body with light.

[0039] As another embodiment of this disclosure, the wavelength of the light can be from 401 nm to 480 nm.

[0040] Additionally, this disclosure provides a method for delivering a target protein into a cell, including a method for administering a composition comprising an efflux body containing a fusion protein to a subject, the fusion protein comprising the target protein and mMaple3.

[0041] Furthermore, this disclosure provides the use of compositions comprising an efflux body containing a fusion protein for delivering a target protein into cells, the fusion protein comprising the target protein and mMaple3.

[0042] In addition, this disclosure provides the use of efflux bodies comprising a fusion protein containing a target protein and mMaple3 for producing formulations for delivering the target protein into cells.

[0043] Technical effect

[0044] The ectosomes according to this disclosure comprise a fusion protein containing blue fluorescent protein (TagBFP), a photocleavable protein (mMaple3), and an ectosome-specific marker protein (CD9). It has been determined that when the ectosome is irradiated with 405 nm light, the photocleavable protein mMaple3 is cleaved, and thus the blue fluorescent protein in the ectosome can be delivered to target cells. Furthermore, it has been determined that when the ectosome containing the Cre fusion protein (Cre-mMaple3-CD9) is irradiated with 405 nm light, the Cre protein in the ectosome can be delivered to animal organs. Therefore, the ectosomes containing the photocleavable protein according to this disclosure are expected to be effectively used in the field of protein therapy by safely and efficiently delivering a variety of therapeutic proteins into cells. Attached Figure Description

[0045] Figure 1 The structure and characteristics of a fusion protein comprising a target protein (blue fluorescent protein), a photocleavable protein, and an ectosome-specific marker protein, according to one embodiment of the present disclosure, are illustrated schematically.

[0046] Figure 2 The preparation process of a fusion protein (TagBFP-mMaple3-CD9) according to one embodiment of the present disclosure and the results of determining its expression in cells are shown.

[0047] Figure 3a The CIBN-CRY2 system is briefly and schematically illustrated.

[0048] Figure 3b A simplified schematic illustration of an implementation of the mMaple system according to this disclosure is provided.

[0049] Figure 4aThe results were obtained by using confocal microscopy to determine the cleavage effect of photoirradiation on the fusion protein (TagBFP-mMaple3-CD9) in HEK293T cells according to one embodiment of the present disclosure.

[0050] Figure 4b The above is illustrated graphically. Figure 4a The fluorescence intensity is shown in the figure.

[0051] Figure 4c The results were obtained by Western blotting in HEK293T cells, which determined the cleavage effect of photoirradiation on the fusion protein (TagBFP-mMaple3-CD9) according to one embodiment of the present disclosure.

[0052] Figure 5 The separation and purification process of effluxes according to one embodiment of the present disclosure is illustrated schematically.

[0053] Figure 6a The results were obtained by measuring the size of the ectosome containing the fusion protein (TagBFP-mMaple3-CD9) using NTA according to one embodiment of this disclosure.

[0054] Figure 6b The results were obtained by measuring the size of the ectosome containing the fusion protein (TagBFP-mMaple3-CD9) using DLS according to one embodiment of this disclosure.

[0055] Figure 6c The results were obtained by measuring the concentration of the efflux body containing the fusion protein (TagBFP-mMaple3-CD9) using microBCA according to one embodiment of this disclosure.

[0056] Figure 6d The result is based on an embodiment of this disclosure, which measures the zeta potential of an efflux body containing the fusion protein (TagBFP-mMaple3-CD9).

[0057] Figure 6e The results are obtained from cryo-electron microscopy (Cryo-TEM) images of an efflux strain containing the fusion protein (TagBFP-mMaple3-CD9) according to one embodiment of this disclosure.

[0058] Figure 6f The results of Western blot analysis, based on one embodiment of this disclosure, determined the cleavage effect of photoirradiation on the fusion protein (TagBFP-mMaple3-CD9) in the ectosome.

[0059] Figure 6gAccording to one embodiment of this disclosure, fluorescence intensity measurements were used to determine the cleavage effect of the fusion protein (TagBFP-mMaple3-CD9) in the ectosome by light irradiation and to determine whether blue fluorescent protein was included in the ectosome.

[0060] Figure 7 The determination of whether the target protein is cleaved is based on the presence or absence of treatment of the ectosome containing the fusion protein (TagBFP-mMaple3-CD9) with Triton X-100, protease (proteinase K), and light according to one embodiment of this disclosure.

[0061] Figure 8a and 8b The results of one embodiment of this disclosure determined the effectiveness of blue fluorescent protein delivery from efflux cells into cells.

[0062] Figure 9 This is the result of determining the effect of delivering the Cre protein in the ectosome to the animal organs when an ectosome containing the fusion protein (Cre-mMaple3-CD9) is administered to an animal according to one embodiment of this disclosure. Detailed Implementation

[0063] This disclosure provides an ectosome containing a fusion protein comprising a target protein and mMaple3.

[0064] Additionally, this disclosure provides compositions for delivering target proteins into cells, comprising efflux bodies as active ingredients.

[0065] In addition, this disclosure provides a method for preparing fusion proteins, which includes the following steps:

[0066] (S1) Amplify the cDNA of the target protein and mMaple3 respectively;

[0067] (S2) The amplified cDNAs of the target protein and mMaple3 are combined into a single cDNA to prepare a cDNA encoding a fusion protein containing the target protein and mMaple3; and

[0068] (S3) The fusion protein is expressed by introducing the cDNA encoding the fusion protein into a vector and then transfecting it into cells.

[0069] In addition, this disclosure provides a method for preparing efflux bodies, which includes the following steps:

[0070] (S1) Amplify the cDNA of the target protein and mMaple3 respectively;

[0071] (S2) The amplified cDNAs of the target protein and mMaple3 are combined into a single cDNA to prepare a cDNA encoding a fusion protein containing the target protein and mMaple3; and

[0072] (S3) The cDNA encoding the fusion protein was transfected into effusor-producing cells, and the effusors were isolated and purified from the cell culture medium.

[0073] In this disclosure, the method may include: combining the amplified cDNA of mMaple3 and the cDNA of an ectosome-specific marker protein into a single cDNA before combining the target protein and the amplified cDNA of mMaple3 into a single cDNA in (S2).

[0074] In this disclosure, (S3) may involve transfecting cDNA encoding the fusion protein into efflux-producing cells, replacing the cell culture medium with FBS-free DMEM medium containing penicillin / streptomycin, and subsequently collecting the culture medium and isolating and purifying the efflux from the collected culture medium.

[0075] In this disclosure, the separation of ectosomes in (S3) may be performed using a method selected from, but not limited to, TFF (tangential flow filtration), ultracentrifugation, size exclusion chromatography, and ectosome separation kits.

[0076] In this disclosure, a "fusion protein" is a protein formed by the fusion of two or more proteins and may include a target protein and mMaple3, and may also include an ectosome-specific marker protein. The target protein, mMaple3, and ectosome-specific marker protein constituting the fusion protein may be combined into one unit, and for example, it may be in the order of target protein-mMaple3-ectosome-specific marker protein, and when the target protein needs to be tracked, it may be in the order of ectosome-specific marker protein-mMaple3-target protein, but there is no limitation on their binding order.

[0077] In this disclosure, "target protein" refers to a protein present in an efflux molecule in combination with a photocleavable protein, and is intended to be delivered to a target cell or tissue. Target proteins can be proteins used to treat or diagnose diseases, and there are no restrictions on their type. For example, the cancer suppressor protein p53 can be delivered to cancer cells as a target protein and thus exhibit therapeutic effects on cancer; it can also exhibit therapeutic effects on Parkinson's disease by delivering normal parkin protein to neurons in Parkinson's disease caused by abnormal function due to mutations in the parkin protein; and in next-generation stem cell therapy research, Oct4, Sox2, c-Myc, and Klf4 proteins, as Yamanica factors, are proteins required for the most important process (the dedifferentiation of patient somatic cells into stem cells) and can thus dedifferentiate into stem cells without the need for viruses currently used for stem cell dedifferentiation. Furthermore, therapeutic effects on degenerative myopathy (e.g., muscular dystrophy characterized by progressive muscle weakness, atrophy, and myofibrosis, etc.) have been demonstrated by delivering myodifferentiation-inducing proteins (e.g., myocyte-derived proteins and MyoD) as target proteins; therapeutic effects on dementia have been demonstrated by delivering NRF2 and BDNF proteins, known for their neuroprotective effects in dementia, one of the neurodegenerative diseases; and therapeutic effects on metabolic diseases have been demonstrated by delivering brown adipose-derived factors (e.g., PGC1α, PPAR-γ) that differentiate white adipocytes into brown adipocytes. In one embodiment of this disclosure, delivery to cells was identified using the blue fluorescent protein TagBFP, and delivery to animal organs was identified using the Cre protein.

[0078] In this disclosure, "delivering a target protein into a cell" means transferring a target protein, which exists in an ectosome in combination with a photocleavable protein, from the ectosome into the target cell.

[0079] In this disclosure, “mMaple3” is a photocleavable protein, and “photocleavable protein” means a protein that is cleaved when exposed to light of a specific wavelength.

[0080] In this disclosure, mMaple3 may contain the amino acid sequence of SEQ ID NO:1 and may be encoded by a gene containing the nucleotide sequence of SEQ ID NO:2.

[0081] In this disclosure, an "exosome-specific marker protein" is a protein located on the outer membrane of an ectosome, and for example, it may be one or more selected from CD9, CD63 and CD81, and according to one embodiment of this disclosure, it may be CD9, but is not limited thereto.

[0082] In this disclosure, CD9 may comprise the amino acid sequence of SEQ ID NO:3 and may be encoded by a gene comprising the nucleotide sequence of SEQ ID NO:4.

[0083] In this disclosure, an "exosome" is a membrane vesicle secreted outside the cell, possessing a lipid bilayer, serving as a collection point for proteins, DNA, RNA, etc., and used for intercellular signal transduction, and is present in the body fluids of almost all eukaryotes. The diameter of an exosome can range from 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, 10 nm to 250 nm, 10 nm to 200 nm, 10 nm to 150 nm, 50 nm to 350 nm, 50 nm to 300 nm, 50 nm to 250 nm, 50 nm to 200 nm, 50 nm to 150 nm, 100 nm to 300 nm, 100 nm to 200 nm, or 100 nm to 150 nm, and is released from the cell when multiple vesicles fuse with the cell membrane, or is released immediately from the cell membrane.

[0084] The ectosome can be prepared using methods known in the art for extracting ectosomes, and there are no limitations on the extraction method.

[0085] Additionally, this disclosure provides a method for delivering a target protein into cells in vitro, comprising irradiating an efflux body containing a fusion protein comprising the target protein and mMaple3 with light; and

[0086] Target cells were treated with efflux bodies that had been irradiated by light.

[0087] Additionally, this disclosure provides a method for screening protein drugs for delivery to cells, comprising (a) irradiating an efflux body containing a fusion protein comprising a protein candidate drug and mMaple3 with light.

[0088] (b) Treatment of target cells with light-irradiated ectosomes; and

[0089] (c) If mMaple3 exhibits fluorescence in target cells, the protein candidate drug is identified as a protein drug to be delivered to the cells.

[0090] In this disclosure, mMaple3 in the ectosome can be cleaved by irradiating the ectosome with light, and subsequently, the wavelength of the light can be 401 nm to 480 nm, 401 nm to 470 nm, 401 nm to 460 nm, 401 nm to 450 nm, 401 nm to 440 nm, 401 nm to 430 nm, 401 nm to 420 nm, or 401 nm to 410 nm, and light of 400 nm or less is ultraviolet (UV) light and can damage cells or ectosomes when treated, and light with wavelengths exceeding 480 nm can also affect cells when treated. In this disclosure, there is no limitation on the wavelength of the light, as long as it is within the range of 401 nm to 480 nm; however, according to one embodiment of this disclosure, preferably, it can be 405 nm.

[0091] In this disclosure, in (c), mMaple3 exhibits green fluorescence when uncut and red fluorescence when cut.

[0092] Additionally, this disclosure provides a method for delivering a target protein into a cell, comprising applying a composition containing an efflux body of a fusion protein to a subject, the fusion protein comprising the target protein and mMaple3.

[0093] In this disclosure, “object” means an object that requires the target protein, and more specifically, it means a human or non-human primate, or a mammal such as a mouse, dog, cat, horse, or cow.

[0094] In this disclosure, “application” means supplying the specified composition of this disclosure to the subject by any suitable method.

[0095] Furthermore, this disclosure provides the use of compositions comprising an efflux body containing a fusion protein for delivering a target protein into cells, said fusion protein containing the target protein and mMaple3.

[0096] Additionally, this disclosure provides the use of efflux bodies comprising a fusion protein containing a target protein and mMaple3 for producing formulations for delivering the target protein into cells.

[0097] In one embodiment of this disclosure, a fusion protein (TagBFP-mMaple3-CD9) comprising a combination of blue fluorescent protein (TagBFP), a photocleavable protein (mMaple3), and an ectosome-specific marker protein (CD9) was prepared, and an advantage was shown when using mMaple3 compared to other photocleavable protein types, and the advantage shown by the mMaple system compared to the CIBN-CRY2 system was determined (see Example 1).

[0098] In another embodiment of this disclosure, it was determined that when the fusion protein (TagBFP-mMaple3-CD9) was overexpressed in HEK293T cells and subsequently irradiated with 405 nm light, mMaple3 was cleaved by 405 nm light (see Example 2).

[0099] In another embodiment of this disclosure, it was determined that mMaple3 was optically cleaved at 405 nm when the efflux protein (TagBFP-mMaple3-CD9) was isolated and purified from the culture medium of HEK293T cells in which it was overexpressed (see Example 3).

[0100] In another embodiment of this disclosure, as a result of determining target protein degradation based on the presence or absence of treatment of the ectosome containing the fusion protein (TagBFP-mMaple3-CD9) with Triton X-100, protease (proteinase K), and 405 nm light, it was determined that the target protein was not degraded by the protease, and that the 405 nm light did not affect the lipid bilayer of the ectosome unless it was artificially permeable to the lipid bilayer of the ectosome by Triton X-100 treatment (see Example 4).

[0101] In one experimental example of this disclosure, it was determined that when an ectosome containing the fusion protein (TagBFP-mMaple3-CD9) was irradiated with 405 nm light and HEK293T cells were treated with it, blue fluorescent protein was delivered into the cells (see Experimental Example 1).

[0102] In one experimental example of this disclosure, it was determined that when an ectosome containing the Cre fusion protein (Cre-mMaple3-CD9) was irradiated with light at 405 nm and administered to genetically modified mice, the Cre protein in the ectosome was delivered to mouse organs, wherein red fluorescent protein (tdTomato) was expressed when the Cre protein was delivered (see Experimental Example 2).

[0103] In the following sections, preferred embodiments and experimental examples are presented to aid in understanding this disclosure. However, these embodiments and examples are provided merely to facilitate a better understanding of this disclosure, and the content of this disclosure is not limited to these embodiments and examples.

[0104] Example 1. Preparation of fusion proteins and comparison of characteristics of photocleavable proteins

[0105] 1-1. Preparation of fusion proteins

[0106] A cDNA encoding a fusion protein (TagBFP-mMaple3-CD9) comprising a combination of blue fluorescent protein (TagBFP), photocleavable protein (mMaple3), and ectosome-specific marker protein (CD9) was prepared, and the structure and characteristics of the fusion protein translated from the prepared cDNA are schematically shown in [the diagram]. Figure 1 The amino acid sequences of TagBFP, mMaple3, and CD9, and the gene sequences encoding them, are shown in Table 1 below.

[0107] [Table 1]

[0108]

[0109]

[0110]

[0111] Specifically, cDNAs encoding TagBFP, mMaple3, and CD9 were first prepared and amplified by PCR. The amplified mMaple3 and CD9 cDNAs were then combined into a single cDNA (mMaple3-CD9) by PCR. Next, the mMaple3-CD9 cDNA and TagBFP cDNA were combined into a single cDNA (TagBFP-mMaple3-CD9) by PCR to generate the cDNA encoding the fusion protein. The primers used in this disclosure are shown in Table 2 below.

[0112] [Table 2]

[0113]

[0114] Subsequently, the cDNA encoding the fusion protein was cloned into the pCMV14 vector using EcoR1 and Xba1 restriction enzymes, enabling the fusion protein to be expressed in mammalian cells. When pCMV14-TagBFP-mMaple3-CD9 was transfected into HEK293T cells using PEI (polyethyleneimine), as... Figure 2 As shown in the figure, the expression of the fusion protein was determined.

[0115] 1-2. Comparison based on the characteristics of photolytically cleavable protein types

[0116] By referring to conventional literature (S. Wang, et al., Proc. Natl. Acad. Sci. USA 111, 8452-8457 (2014)), and comparing the characteristics of various photocleavable proteins (PAFPs) (e.g., Dendra2, mEos2, tdEos, mKikGR, and Kaede) with mMaple3, it was determined that mMaple3 exhibits an advantage compared to other photocleavable proteins.

[0117] As a result, as shown in Table 3 below, it was determined that many photocleavable proteins generally have a strong tendency to form oligomers in terms of oligomerization and cohesion, however mMaple3 has a strong tendency to retain monomers and weak cohesion.

[0118] Regarding the Switchλ results showing the wavelengths of light used for cleavage, it was determined that KikGR and Kaede require light of 400 nm or less to be cleaved, while 400 nm or less is UV (ultraviolet light) and can damage cells or ectosomes when treated. However, it is safer to be cleaved after intracellular expression or by addition to ectosomes when irradiated with light of 400 nm or greater, and mMaple3 is cleaved under light of 400 nm or greater.

[0119] Among photocleavable proteins expressed in cells, there may be photocleavable proteins that are not fully folded until fluorescence is measured. Among the fully folded photocleavable proteins, only some are photocleaved and show fluorescence. It can be determined that mMaple3 is cleaved more efficiently and shows fluorescence than other photocleavable proteins due to its high localization number / cell ratio, which indicates the number of PAFPs (photoactivated fluorescent proteins) detected by fluorescence / cell / actual PAFP expression level.

[0120] In addition, although most photocleavable proteins are acid-sensitive, no acid sensitivity has been reported in the case of mMaple3.

[0121] [Table 3]

[0122] PAFP Oligomerization Gathering Switchλ(nm) Number of localizations / cells acid sensitivity Dendra2 monomer - 405.480 1,810 high mEos2 weak dimer + 405 1,290 medium tdEos Tetramer - 405 1,800 NA mKikGR monomer + 390 3,800 high Kaede Tetramer + 380 NA medium mMaple3 monomer - 405 12,300 NA

[0123] 1-3. Comparison of CIBN-CRY2 system and mMaple system

[0124] Figure 3aThe CIBN-CRY2 system shown uses two light-specific binding proteins, therefore two constructs should always be used, and in this case, efficiency is reduced because both constructs should be co-transfected. For example, when the transfection efficiency is 80%, the probability of all two constructs being transfected into one cell becomes 64%. On the other hand, Figure 3b The mMaple system shown uses a construct that allows it to be expressed more efficiently in cells.

[0125] In addition, the CIBN-CRY2 system has the disadvantage that it can affect cells because 488 nm light must be continuously applied to cells during ectosome formation, and the amount of carrier protein contained in the ectosome is lost because the binding efficiency of light to CIBN and CRY2 cannot reach 100%. However, the mMaple system is safe because the carrier protein is fused with photocleavable proteins and ectosome-specific markers at the beginning, so the carrier protein is not lost when the cell forms the ectosome, and there is no need to apply light directly to the cell.

[0126] Furthermore, in the CIBN-CRY2 system, there may be instances where CIBN and CRY2 binding cannot be maintained under light-free conditions after the formation of the ectosome. In such cases, the carrier protein cannot move freely and can bind to ectosome-specific proteins. However, the mMaple system cleaves photocleavable proteins by applying light to the ectosome after its formation, and the cleavage efficiency is very high. When the cleavage efficiency is high, it means that many freely moving carrier proteins are generated in the ectosome.

[0127] Furthermore, the CIBN-CRY2 system has the disadvantage that the large size of CRY2 (65 kDa) can affect the intrinsic function of the carrier protein, fluorescent proteins (such as EGFP) should be fused and used separately to determine whether they are well expressed in the cell, and there is no way to determine whether the binding of CRY2 and CIBN is disrupted when no light is applied. In contrast, the mMaple system has a very low probability of affecting the intrinsic function of the carrier protein because the photocleavable protein fragment linked to the carrier protein is only 10 kDa in size after cleavage. Since the photocleavable protein itself is a fluorescent protein, its proper expression in the cell can be determined by green fluorescence, and the good delivery of the photocleavable protein can be determined by red fluorescence after light treatment of the ectosome.

[0128] As described above, compared to other types of photocleavable proteins and light-specific binding proteins containing the CIBN-CRY2 system, the fusion protein containing the photocleavable protein mMaple3 according to this disclosure exhibits the advantages shown in Examples 1-2 and 1-3 above, and accordingly, the effectiveness of protein delivery into cells was determined through experiments using the fusion protein containing the photocleavable protein mMaple3 according to this disclosure.

[0129] Example 2. Determination of fusion protein delivery

[0130] Twenty-four hours after inoculation of HEK293T cells, TagBFP-mMaple3-CD9 cDNA was transfected with PEI using the method described in Example 1-1 above to overexpress it in HEK293T cells. The photocleavage effect was then determined in HEK293T cells by irradiation with 405 nm light, confocal microscopy, and Western blot.

[0131] Experimental results observed by confocal microscopy confirmed that the expression of the fusion protein was obtained 24 hours after transfection using a confocal microscope (LSM700) via blue fluorescence (TagBFP) and green fluorescence (mMaple3). Figure 4a (in Chinese), and the delivery of the fusion protein was determined by the decreased green fluorescence intensity (mMaple3 before cleavage) and the increased red fluorescence intensity (mMaple3 after cleavage). The fluorescence intensity was measured and shown in [the image / image / etc.]. Figure 4b middle.

[0132] As a result, such Figure 4a and 4b As shown, given that mMaple3 exhibits green fluorescence before irradiation with 405 nm light and red fluorescence after irradiation with 405 nm light, it can be determined that the mMaple3 protein is cleaved. Furthermore, it can be determined that the longer the irradiation time with 405 nm light, the more photo-cleavable protein (mMaple3) is cleaved, thus reducing green fluorescence and increasing red fluorescence.

[0133] Additionally, 24 hours post-transfection, cells were irradiated with 405 nm light for 5 minutes. Western blotting was used, proteins were extracted with T-per buffer, and the extracted proteins were separated by electrophoresis. Subsequently, the expression of the fusion protein and the cleavage effect of 405 nm light on the fusion protein were determined. As a result, such as... Figure 4c As shown, the cleavage effect on mMaple3 protein was determined in HEK293T cells by observing a decrease in the amount of uncleaved fusion protein and an increase in the amount of cleaved fusion protein segments over time after irradiation with 405 nm light.

[0134] Example 3. Efflux body separation and purification

[0135] Efflux cells were separated and purified from the culture medium of HEK293T cells overexpressing the fusion protein in Example 2 above using a tangential fluid filtration system.

[0136] Specifically, 24 hours after inoculation of HEK293T cells, TagBFP-mMaple3-CD9 cDNA was transfected using PEI, and 24 hours after transfection, the cell culture medium was replaced with FBS-free DMEM (1% PS (penicillin / streptomycin)). 24 hours after the medium replacement, the culture medium was collected for the first time, and FBS-free DMEM (1% PS) medium was added again, and a second collection was made 24 hours later. Effelosomes were isolated and purified from the first and second collections of culture medium using TFF (tangential flow filtration). The isolation and purification process of the effelosomes is schematically shown in [illustration missing]. Figure 5 middle.

[0137] After isolating and purifying the effluxes using this method, the concentration and size of the effluxes containing the fusion protein were measured using NTA (Nanoparticle Tracking Assay), DLS (Dynamic Light Scattering), and microBCA (Bicinchoninic Acid Assay).

[0138] As a result of NTA measurement, such as Figure 6a As shown in the figure, the average diameter of the extravasated body is 132 nm; as a result of DLS measurement, such as Figure 6b As shown, the average diameter of the extravasated body was determined to be 117.7 ± 10.28 nm; and as a result of microBCA measurements, such as Figure 6c As shown, the protein concentration of the ectosome containing the fusion protein is 1487 mg / mL, and the amount of protein contained in one ectosome is determined to be 39.13 ng.

[0139] Furthermore, as a result of measuring the zeta potential of the efflux body, such as Figure 6d As shown, it was determined to be an average of -19.3 ± 0.8 mV, and the cryo-electron microscopy (Cryo-TEM) image observation data of the efflux body are shown in... Figure 6e Medium (scale bar: 100nm).

[0140] In addition, as a result of determining the cleavage effect of 405 nm light on the fusion protein in the ectosome using Western blotting, such as Figure 6fAs shown, the cleavage effect on mMaple3 protein was determined in ectosomes by observing a decrease in the amount of uncleaved fusion protein and an increase in the amount of cleaved fusion protein segments over time after irradiation with 405 nm light.

[0141] As a result of measuring the intensity of blue, green, and red fluorescence using a BioTek microplate reader to determine the cleavage effect of 405nm light on the fusion protein and whether the efflux protein is present again in the efflux body, such as... Figure 6g As shown, it was determined that there was no difference in blue fluorescence intensity before and after irradiation with 405 nm light, and it was observed that the green fluorescence intensity decreased significantly after irradiation with 405 nm light, while the red fluorescence intensity increased. This determined the cleavage effect of the fusion protein in the exosome and whether the exosome contained blue fluorescent protein.

[0142] Example 4. Determination of target protein location in efflux bodies

[0143] To determine the location of the target protein (TagBFP) in the ectosome (+mMaple3 ectosome) containing the fusion protein (TagBFP-mMaple3-CD9) isolated in Example 3 above, a protease digestion assay was performed.

[0144] Specifically, the degradation of the target protein was determined by treatment with a protease (proteinase K) and Triton X-100 that enable it to pass through the efflux lipid bilayer.

[0145] As a result, such Figure 7 As shown, it can be observed that unless the lipid bilayer of the ectosome is artificially permeated using Triton X-100, the protein (ALIX) containing the target protein (TagBFP) inside the ectosome is not degraded and is well protected from proteases (proteinase K). On the other hand, it was determined that the protein with the external domain of the ectosome (LAMP2) is degraded by proteases regardless of whether Triton X-100 treatment is used. Furthermore, it was determined that 405 nm light does not affect the lipid bilayer of this ectosome.

[0146] Experimental Example 1. Determining the entry of proteins from effelosomes into cells

[0147] For the ectosomes containing the fusion protein (TagBFP-mMaple3-CD9) isolated in Example 3 above (+mMaple3 ectosomes), ectosomes without specific proteins isolated and purified from HEK293T cell culture solution (+negative ectosomes) were used as a control group, and HEK293T cells were treated with 405nm light.

[0148] Specifically, within 24 hours after HEK293T cell inoculation, at a dose of 5 × 10⁻⁶ cells / mL... 9 Particle / mL concentrations were obtained by treating ectosomes containing the TagBFP-mMaple3-CD9 fusion protein, and HEK293T cells were fixed with 4% paraformaldehyde within 24 hours after ectosome treatment. Delivery of blue fluorescent protein was then determined using a Cytation 5 cell imaging system.

[0149] As a result, such Figure 8a As shown, it was observed that when treated with ectosomes containing mMaple3, blue fluorescence (TagBFP) was observed in the cells, and it was determined that when the ectosomes were treated with light, green fluorescence (FL mMaple3) decreased while red fluorescence (CL mMaple3) increased.

[0150] Additionally, as a result of determining the delivery of blue fluorescent protein in HeLa cells after treatment with ectosomes using the same method, such as Figure 8b As shown, it was determined that blue fluorescence was also observed in HeLa cells, and that green fluorescence decreased while red fluorescence increased when the efflux was treated with light.

[0151] This confirms that in the ectosome containing TagBFP-mMaple3-CD9, mMaple3 is cleaved by 405 nm light treatment, thereby delivering blue fluorescent protein (TagBFP) into the cell.

[0152] Experimental Example 2. Determination of protein delivery from ectosomes to animal organs

[0153] The ectosome containing Cre fusion protein (Cre-mMaple3-CD9) isolated by the method described in Example 3 above was treated with 405 nm light, and when it was administered to genetically modified mice, the Cre protein was delivered, in which red fluorescent protein (tdTomato) was expressed.

[0154] Specifically, phosphate-buffered saline (PBS) or phototreated efflux (Cre:MAPLEX) was administered at 500 μg to the tail vein of genetically modified mice, and the intensity of red fluorescence was subsequently measured using an IVIS small animal imaging system at 1, 6, and 24 hours later.

[0155] As a result, such Figure 9 As shown, it was determined that the intensity of red fluorescence was increased in the livers of mice treated with phototreated efflux bodies, such as... Figure 9As shown in the figure. It can be determined that in the ectosome containing Cre-mMaple3-CD9, mMaple is cleaved by light treatment at 405 nm, thereby efficiently delivering the Cre protein into the organ.

[0156] The foregoing description of this disclosure is for illustrative purposes only, and those skilled in the art will understand that it can be readily modified into other specific forms without altering the technical spirit or essential characteristics of this disclosure. Therefore, it should be understood that the above embodiments are illustrative in all respects and are not restrictive.

[0157] Industrial applicability

[0158] It is anticipated that the efflux bodies containing photocleavable proteins disclosed herein could be effectively used in the field of protein therapy by safely and efficiently delivering a variety of therapeutic proteins into cells. sequence list <110> RESEARCH & BUSINESS FOUNDATION SUNGKYUNKWAN UNIVERSITY <120> Evosomes containing photocleavable proteins and their uses <130> MPCT21-003 <150> KR 10-2020-0015696 <151> 2020-02-10 <150> KR 10-2021-0015837 <151> 2021-02-04 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 237 <212> PRT <213> Artificial sequence <220> <223> mMaple3 <400> 1 Met Val Ser Lys Gly Glu Glu Thr Ile Met Ser Val Ile Lys Pro Asp 1 5 10 15 Met Lys Ile Lys Leu Arg Met Glu Gly Asn Val Asn Gly His Ala Phe 20 25 30 Val Ile Glu Gly Glu Gly Ser Gly Lys Pro Phe Glu Gly Ile Gln Thr 35 40 45 Ile Asp Leu Glu Val Lys Glu Gly Ala Pro Leu Pro Phe Ala Tyr Asp 50 55 60 Ile Leu Thr Thr Ala Phe His Tyr Gly Asn Arg Val Phe Thr Lys Tyr 65 70 75 80 Pro Arg Lys Ile Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Tyr 85 90 95 Ser Trp Glu Arg Ser Met Thr Tyr Glu Asp Gly Gly Ile Cys Asn Ala 100 105 110 Thr Asn Asp Ile Thr Met Glu Glu Asp Ser Phe Ile Asn Lys Ile His 115 120 125 Phe Lys Gly Thr Asn Phe Pro Pro Asn Gly Pro Val Met Gln Lys Arg 130 135 140 Thr Val Gly Trp Glu Val Ser Thr Glu Lys Met Tyr Val Arg Asp Gly 145 150 155 160 Val Leu Lys Gly Asp Val Lys Met Lys Leu Leu Leu Lys Gly Gly Ser 165 170 175 His Tyr Arg Cys Asp Phe Arg Thr Thr Tyr Lys Val Lys Gln Lys Ala 180 185 190 Val Lys Leu Pro Lys Ala His Phe Val Asp His Arg Ile Glu Ile Leu 195 200 205 Ser His Asp Lys Asp Tyr Asn Lys Val Lys Leu Tyr Glu His Ala Val 210 215 220 Ala Arg Asn Ser Thr Asp Ser Met Asp Glu Leu Tyr Lys 225 230 235 <210> 2 <211> 711 <212> DNA <213> Artificial Sequence <220> <223> mMaple3 <400> 2 atggtgagca aaggcgagga gacaatcatg tccgtgatca agcccgacat gaagatcaaa 60 ctgaggatgg agggcaacgt gaacggccac gccttcgtga tcgagggcga aggaagcggc 120 aagcccttcg agggcatcca gaccatcgat ctggaggtca aggagggcgc tcccctccct 180 ttcgcctatg acatcctgac caccgccttc cactacggca atagggtgtt caccaagtat 240 cccaggaaga tccccgacta cttcaagcag agcttccctg agggctacag ctgggagagg 300 agcatgacat acgaggacgg cggcatctgc aacgccacca acgacatcac aatggaggag 360 gacagcttca tcaacaagat ccacttcaaa ggcacaaact tcccccccaa tggccccgtg 420 atgcagaaga ggaccgtggg ctgggaggtg agcaccgaga agatgtacgt gagggacggc 480 gtcctgaagg gcgacgtgaa gatgaagctc ctgctcaagg gcggcagcca ctacaggtgc 540 gactttagga ccacctataa ggtgaagcag aaggctgtga agctgcccaa ggcccacttc 600 gtcgaccata ggatcgagat cctgtcccac gacaaggact acaacaaggt caagctgtac 660 gagcacgccg tcgctaggaa cagcaccgac agcatggacg aactctataa 711 <210> 3 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> CD9 <400> 3 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> 4 <211> 684 <212> DNA <213> Artificial sequence <220> <223> CD9 <400> 4 atgccggtca aaggaggcac caagtgcatc aaatacctgc tgttcggatt taacttcatc 60 ttctggcttg ccgggattgc tgtccttgcc attggactat ggctccgatt cgactctcag 120 accaagagca tcttcgagca agaaactaat aataataatt ccagcttcta cacaggagtc 180 tatattctga tcggagccgg cgccctcatg atgctggtgg gcttcctggg ctgctgcggg 240 gctgtgcagg agtcccagtg catgctggga ctgttcttcg gcttcctctt ggtgatattc 300 gccattgaaa tagctgcggc catctgggga tattcccaca aggatgaggt gattaaggaa 360 gtccaggagt tttacaagga cacctacaac aagctgaaaa ccaaggatga gccccagcgg 420 gaaacgctga aagccatcca ctatgcgttg aactgctgtg gtttggctgg gggcgtggaa 480 cagtttatct cagacatctg ccccaagaag gacgtactcg aaaccttcac cgtgaagtcc 540 tgtcctgatg ccatcaaaga ggtcttcgac aataaattcc acatcatcgg cgcagtgggc 600 atcggcattg ccgtggtcat gatatttggc atgatcttca gtatgatctt gtgctgtgct 660 atccgcagga accgcgagat ggtc 684 <210> 5 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> TagBFP <400> 5 Met Ser Glu Leu Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu 1 5 10 15 Gly Thr Val Asp Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly 20 25 30 Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly 35 40 45 Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Leu Tyr 50 55 60 Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe 65 70 75 80 Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr 85 90 95 Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp 100 105 110 Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Thr Ser 115 120 125 Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Phe Thr 130 135 140 Glu Thr Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Asn Asp Met 145 150 155 160 Ala Leu Lys Leu Val Gly Gly Ser His Leu Ile Ala Asn Ile Lys Thr 165 170 175 Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val 180 185 190 Tyr Tyr Val Asp Tyr Arg Leu Glu Arg Ile Lys Glu Ala Asn Asn Glu 195 200 205 Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu 210 215 220 Pro Ser Lys Leu Gly His Lys Leu Asn 225 230 <210> 6 <211> 699 <212> DNA <213> Artificial sequence <220> <223> TagBFP <400> 6 atgagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac 60 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc 120 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact 180 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc 240 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc 300 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag 360 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg 420 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg 480 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc 540 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa 600 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga 660 tactgcgacc tccctagcaa actggggcac aagcttaat 699 <210> 7 <211> 27 <212> DNA <213> Artificial sequence <220> <223> TagBFP - Forward Primer for Connector <400> 7 tatgctgaat tcgccaccat gagcgag 27 <210> 8 <211> 52 <212> DNA <213> Artificial sequence <220> <223> TagBFP - Connector Reverse Primer <400> 8 ggaagcttga gctcgagatc tgagtccgga attaagcttg tgccccagtt tg 52 <210> 9 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Connector-mMaple3-Connector forward primer <400> 9 tctcgagctc aagcttccgt gagcaaaggc gaggag 36 <210> 10 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Connector-mMaple3-Connector Reverse Primer <400> 10 acctccgcct gaaccgccac ctcccgactt atagagttcg tccatgctgt c 51 <210> 11 <211> 56 <212> DNA <213> Artificial sequence <220> <223> Connector-CD9 forward primer <400> 11 ggcggttcag gcggaggtgg ctctggcggt ggcggatcgc cggtcaaagg aggcac 56 <210> 12 <211> 31 <212> DNA <213> Artificial sequence <220> <223> Connector-CD9 reverse primer <400> 12 ccctctagtc tagagaccat ctcgcggttc c 31

Claims

1. Ectosome, which contains a fusion protein of the target protein - mMaple3-CD9; in: The mMaple3 consists of the amino acid sequence of SEQ ID NO: 1; and The CD9 consists of the amino acid sequence of SEQ ID NO:

3.

2. The effluent body according to claim 1, The mMaple3 is encoded by a gene containing the nucleotide sequence of SEQ ID NO:

2.

3. The effluent body according to claim 1, The target protein is to be delivered into the cell.

4. The effluent body according to claim 3, The target protein is used to treat or diagnose diseases.

5. A composition for delivering a target protein into cells, comprising an ectosome as an active ingredient according to any one of claims 1 to 4.

6. The composition according to claim 5, The target protein is to be delivered into the cell.

7. The composition according to claim 6, The target protein is used to treat or diagnose diseases.

8. A method for preparing the fusion protein of claim 1, comprising the following: (S1) Amplify the cDNA of CD9, target protein and mMaple3 respectively; (S2) Combining the amplified cDNA of CD9 and mMaple3 with the amplified cDNA of the target protein into a single strand to prepare a cDNA encoding a fusion protein comprising CD9, the target protein, and mMaple3; and (S3) The fusion protein is expressed by introducing the cDNA encoding the fusion protein into a vector and then transfecting it into cells.

9. The method of claim 8, wherein the method further comprises: Before combining the amplified cDNA of mMaple3 with the amplified cDNA of the target protein into a single strand in (S2), the amplified cDNA of mMaple3 is combined with the cDNA of CD9 into a single strand.

10. A method for preparing the efflux body according to claim 1, comprising the following: (S1) Amplify the cDNA of CD9, target protein and mMaple3 respectively; (S2) Combine the amplified cDNA of CD9 and mMaple3 with the amplified cDNA of the target protein into a single strand to prepare a cDNA encoding a fusion protein containing CD9, the target protein and mMaple3. as well as (S3) The cDNA encoding the fusion protein is transfected into effusor-producing cells, and the effusor is isolated and purified from the cell culture medium.

11. The method according to claim 10, The method further includes: Before combining the amplified cDNA of mMaple3 with the amplified cDNA of the target protein into a single strand in (S2), the amplified cDNA of mMaple3 is combined with the cDNA of CD9 into a single strand.

12. The method according to claim 10, In step (S3), the separation of the ectosome is performed by a method selected from the following: tangential flow filtration (TFF), ultracentrifugation, size exclusion chromatography, and a method using an ectosome separation kit.

13. Use of the efflux organism of claim 1 in the preparation of a composition for in vitro delivery of a target protein into cells, wherein the composition is formulated for: Irradiate the efflux body with light; and Treat target cells with light-irradiated ectosomes; The wavelength of the light is between 401 nm and 480 nm.

14. The use according to claim 13, The mMaple3 in the exosome is separated by irradiating the exosome with light.

15. A method for screening protein drugs for delivery into cells, comprising: (a) Irradiating the efflux body of claim 1 with light, wherein the target protein is a protein candidate drug; (b) Treat target cells with light-irradiated ectosomes; as well as (c) If the mMaple3 exhibits fluorescence in the target cells, the protein candidate drug is identified as a protein drug to be delivered to the cells; The wavelength of the light is between 401 nm and 480 nm.

16. The method according to claim 15, When the light is irradiated in step (a), the mMaple3 in the exosome is separated.

17. Use of the efflux body of claim 1 in the preparation of compositions for delivering target proteins into cells in subjects where such delivery is desired.

18. Use of the efflux body of claim 1 for preparing a formulation for delivering the target protein into cells.

Images