A plasmid platform for stable expression and transfer of biomolecules

Abstract

The plasmid platform proposed in the present invention comprises a nucleic acid sequence encoding a modified protein in which the intracellular domain, extracellular domain, or a combination thereof of LAMP-2B (Lysosome-Associated Membrane Glycoprotein 2B) has been deleted, and is based on the discovery that when a specific domain of LAMP-2B is deleted, the expression of the biomolecule to be expressed is significantly stabilized and the delivery efficiency of the biomolecule to be delivered is significantly increased. This is expected to provide high technical utility in the future in the expression and delivery of target proteins, particularly in the expression and delivery using exosomes, and can be expanded to various related technical fields or applications.

Description

[Technical Field]

[0001] The present invention relates to a plasmid platform for stable expression and transfer of biomolecules. [Background technology]

[0002] Exosomes are produced from the budding of late endosomes and fuse with the plasma membrane before being released into the extracellular space. Exosomes are 40–200 nm vesicles composed of a lipid bilayer membrane rich in phosphocholine, cholesterol, and ceramide. They are secreted by almost all types of cells and are stable in all types of body fluids, such as blood, lymph, and sweat. Their small size and weak negative charge allow for long circulation times and the ability to reach organs. Exosomes can also evade phagocytosis, deliver hydrophilic or hydrophobic drugs, and cross the vascular endothelium to target cells. Exosomes are known to target specific cells via specific surface proteins, such as tetraspanins. Exosome encapsulation has been reported to increase the stability and bioavailability of curcumin in vitro and in vivo, as well as its anti-inflammatory activity. Other studies have shown that exosomes can deliver doxorubicin across the blood-brain barrier to the brain, deliver siRNA to cells, and effectively reduce levels of the RAD51 protein, a potent target for cancer treatment. Summary of the Invention [Problem to be solved by the invention]

[0003] One object of the present invention is to provide a plasmid platform for stable expression and delivery of biomolecules, including nucleic acid sequences encoding modified proteins from Lysosome-Associated Membrane Glycoprotein 2B (LAMP-2B) in which the intracellular domain, the extracellular domain, or a combination thereof has been deleted.

[0004] One object of the present invention is to provide a recombinant plasmid for stable expression and transfer of a biomolecule, which further comprises a sequence encoding the biomolecule to be expressed and transferred within said plasmid platform.

[0005] An object of the present invention is to provide an exosome for stable expression and delivery of a biomolecule, comprising said recombinant plasmid.

[0006] An object of the present invention is to provide a composition for diagnosing cancer, which comprises the exosome, and the biomolecule is a peptide that specifically binds to a protein specifically expressed on the surface of cancer cells.

[0007] The technical problems that the present invention aims to achieve are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description. [Means for solving the problem]

[0008] In one aspect, the present invention provides a plasmid platform for stable expression and delivery of biomolecules, including a nucleic acid sequence encoding a modified protein from Lysosome-Associated Membrane Glycoprotein 2B (LAMP-2B) in which the intracellular domain, the extracellular domain, or a combination thereof has been deleted.

[0009] The plasmid platform is based on the discovery that when the previously known intracellular, extracellular, or both of LAMP-2B are removed, the stability of expression of the target protein to be expressed inside or outside the cell together with LAMP-2B is significantly increased, and its transduction into the target cell is also significantly increased.

[0010] The LAMP-2B may refer to a conventionally known amino acid sequence or a nucleic acid sequence encoding the same, and may refer to a nucleic acid sequence to be loaded into a plasmid platform. In a specific embodiment, the LAMP-2B may be a sequence having the accession number NM_013995.2, but is not particularly limited thereto.

[0011] The intracellular domain and extracellular domain may refer to the region expressed inside and outside the phospholipid bilayer of a cell when LAMP-2B is expressed on the cell, respectively. Considering the case where LAMP-2B is expressed on exosomes, the intracellular domain may preferably refer to the region expressed inside the phospholipid bilayer of an exosome, and the extracellular domain may preferably refer to the region expressed outside the phospholipid bilayer of an exosome.

[0012] The modified protein is characterized by having the intracellular domain, the extracellular domain, or a combination thereof of LAMP-2B deleted. Preferably, the intracellular domain may be deleted for the purpose of stabilizing the expression of a biomolecule (or target protein) to be expressed in the extracellular region (or the outer region of the phospholipid bilayer) and a biomolecule (or active protein) to be expressed in the intracellular region (or the inner region of the phospholipid bilayer). In this sense, the plasmid platform may contain a nucleic acid sequence encoding a protein from which the intracellular domain of LAMP-2B has been deleted.

[0013] Furthermore, the modified protein may preferably have both the intracellular and extracellular domains deleted for the purpose of stabilizing the expression of the biomolecule to be expressed in the extracellular region and the biomolecule to be expressed in the intracellular region, while simultaneously stably delivering the biomolecule to the target cell. In this regard, the plasmid platform may have both the intracellular and extracellular domains deleted of LAMP-2B.

[0014] According to one embodiment, the nucleic acid sequence may have any one of the nucleic acid sequences of SEQ ID NOs: 2 to 4, and may preferably have the nucleic acid sequence of SEQ ID NO: 2 or 4 for the purpose of stabilizing the expression of a biomolecule to be expressed in the intracellular region, and more preferably have the nucleic acid sequence of SEQ ID NO: 4 for the purpose of stabilizing the expression of a biomolecule to be expressed in the intracellular region while simultaneously stably transferring the biomolecule to a target cell.

[0015] The plasmid platform may further include a nucleic acid sequence encoding a glycosylated region, which may be for the purpose of stabilizing the expression and delivery of a biomolecule to be expressed by being loaded onto the plasmid platform, more specifically, for the purpose of stabilizing the expression and delivery of all biomolecules in the intracellular region and extracellular region, but is not particularly limited thereto.

[0016] The glycosylated region can be introduced into a protein expression construct using any known method. In terms of application to the plasmid platform, the glycosylated region may be incorporated into the plasmid platform in the form of a nucleic acid sequence encoding the glycosylated region. For example, the plasmid platform may include a nucleic acid sequence encoding a GNS™ motif; a sequence encoding the specific amino acid sequence NXS (where X can be any amino acid sequence except proline), NXT (where X can be any amino acid sequence except proline), or NXC (where X can be any amino acid sequence except proline) that can induce N-linked glycosylation; a sequence encoding 1-5 amino acids at the C-terminus of the N-glycosylated amino acid sequence; a sequence encoding glycine at the N-terminus of the sequence containing 1-5 amino acids at the C-terminus of the N-glycosylated amino acid sequence; or an N-linked glycosylation motif in tetraspanins (CD9 SEL: Small Extracellular Loop Domain, CD63 LEL: Large Extracellular Loop Domain). At least one method can be selected from the group consisting of a method of including a sequence encoding a GNSTM motif in the plasmid platform; and a method of including serine or threonine, which are amino acid sequences that induce O-glycosylation. From the viewpoint of simultaneously maximizing stable expression and transfer efficiency of a biomolecule, the method of including a nucleic acid sequence encoding a GNSTM motif in the plasmid platform is preferred, but is not particularly limited thereto.

[0017] The nucleic acid sequence encoding the glycosylation region is not limited to a particular location as long as it can be included in the plasmid platform. For example, it can be included so that it is located in the direction of the extracellular region (or the outer region of the phospholipid bilayer) relative to the nucleic acid sequence encoding the modified protein. More specifically, a sequence encoding a biomolecule (or target protein) can be located in the direction of the extracellular region of the nucleic acid sequence encoding the modified protein, and the nucleic acid sequence encoding the glycosylation region can be located upstream (or toward the extracellular region) or downstream (or toward the intracellular region) of the sequence encoding the biomolecule, but is not particularly limited thereto. According to a preferred embodiment, the nucleic acid sequence encoding the glycosylation region can be located in the direction of the extracellular region relative to the nucleic acid sequence encoding the modified protein.

[0018] The nucleic acid sequence encoding the glycosylated region may include any one of the nucleic acid sequences of SEQ ID NOs: 11 to 13, and from the viewpoint of simultaneously maximizing stable expression and transfer efficiency of the biomolecule, it may preferably include the nucleic acid sequence of SEQ ID NO: 11.

[0019] The coding sequence of the plasmid platform may be configured to include the glycosylation region and simultaneously have the modified protein form, as described above. According to a preferred embodiment, the plasmid platform may simultaneously include a sequence encoding a GNSTM motif and a sequence encoding a modified protein from which the extracellular and intracellular domains of LAMP-2B have both been deleted. As a more specific example, the sequence encoding the GNSTM motif may be located upstream (or toward the extracellular domain) of the sequence encoding the modified protein. As a further example, the plasmid platform may include the nucleic acid sequence of SEQ ID NO: 4 and the nucleic acid sequence of SEQ ID NO: 11.

[0020] In one aspect of the present invention, there is provided a recombinant plasmid for stable expression and delivery of a biomolecule, which further comprises a nucleic acid sequence encoding the biomolecule to be expressed and delivered within the above-described plasmid platform.

[0021] The nucleic acid sequence encoding the biomolecule is not particularly limited in its location as long as it is located within the above-mentioned plasmid platform and can successfully express the biomolecule of interest. Specific examples include: i) between the nucleic acid sequence encoding the glycosylated region and the nucleic acid sequence encoding the modified protein; ii) toward the intracellular region relative to the nucleic acid sequence encoding the modified protein; or iii) all of these.

[0022] When the nucleic acid sequence encoding the biomolecule is positioned extracellularly relative to the nucleic acid sequence encoding the modified protein, the purpose may be to express or deliver the biomolecule extracellularly (or to the external region of the phospholipid bilayer), and when the nucleic acid sequence encoding the modified protein is positioned intracellularly relative to the nucleic acid sequence encoding the modified protein, the purpose may be to express or deliver the biomolecule intracellularly (or to the internal region of the phospholipid bilayer), but is not particularly limited thereto.

[0023] The biomolecule is not particularly limited as long as it is to be expressed using the plasmid platform, and specific examples thereof include at least one selected from the group consisting of nucleic acid molecules, aptamers, peptides, proteins, glycoproteins, lipoproteins, immunoglobulins, hormones, growth factors, recombinases, and fluorescent proteins.

[0024] In one aspect of the present invention, there is provided an exosome for stable expression and delivery of a biomolecule, comprising a product expressed from the recombinant plasmid described above.

[0025] The exosomes contain modified proteins and biomolecules expressed from the recombinant plasmids described above, and the biomolecules can be located inside, outside, or both of the exosomes, stably expressed, and delivered to cells of interest.

[0026] The biomolecule may be expressed on the outside of the exosome and include a substance that specifically binds to the surface of a target cell. In such cases, the targeting ability of the exosome to the target cell is increased, the expressed biomolecule can be effectively delivered, and the presence or absence of the target cell can be determined by detecting the expressed biomolecule. Furthermore, exosomes can be used for various well-known applications based on target cell targeting, without any particular limitations. For example, if a biomolecule that can specifically bind to a substance specifically expressed on the surface of cells infected with a specific virus or cancer cells is expressed on the outside of the exosome, the exosome can specifically bind to cells infected with a specific virus or cancer cells, and can be used to determine the presence or absence of infection with a specific virus or the presence or absence of cancer cells, while simultaneously delivering the biomolecule contained in the exosome into the virus-infected cells or cancer cells.

[0027] In one aspect, the present invention provides a composition for diagnosing cancer, comprising the exosome described above, wherein the biomolecule comprises one that specifically binds to a protein specifically expressed on the surface of cancer cells.

[0028] In addition, one aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the exosome described above, wherein the biomolecule specifically binds to a protein specifically expressed on the surface of cancer cells, and a therapeutic substance to be delivered into cancer cells.

[0029] The biomolecule may include one that specifically binds to a protein specifically expressed on the surface of cancer cells, and may include one that is expressed in the external bilayer region of the exosome. In such a case, the biomolecule expressed in the external region specifically binds to the surface of cancer cells, thereby targeting the exosome to cancer cells, enabling the exosome to be used for cancer diagnosis based on such targeting.

[0030] Furthermore, the biomolecule may include a therapeutic substance to be delivered into cancer cells, and such a therapeutic substance may include a substance expressed in the inner bilayer region of the exosome. In this case, the exosome can deliver the therapeutic substance into cancer cells through interaction with the cancer cells (e.g., surface receptor interaction, membrane fusion, receptor-mediated endocytosis, phagocytosis, micropinocytosis, etc.), thereby achieving a preventive or therapeutic effect against cancer.

[0031] The cancers include carcinomas, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer, including squamous cell carcinoma; lymphoid hematopoietic malignancies, including leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkitt's lymphoma; myeloid hematopoietic malignancies, including acute and chronic myeloid leukemia and promyelocytic leukemia; fibrosarcoma and striated muscle other tumors including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, and according to a preferred embodiment, may be any one selected from the group consisting of, but not limited to, ovarian cancer.

[0032] The compositions of the present invention may further comprise a pharmaceutically acceptable carrier and can be formulated together with a carrier. In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate living organisms and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated into liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, can be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0033] The compositions of the present invention may be in any dosage form containing the exosomes of the present invention as an active ingredient, and may be prepared in oral or parenteral dosage forms. Pharmaceutical dosage forms of the present invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or forms suitable for administration by inhalation or insufflation.

[0034] The compositions of the present invention are administered in a pharmaceutically effective amount. The effective dose level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, in single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by one skilled in the art.

[0035] The dosage of the composition of the present invention varies widely depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The appropriate dosage may vary depending on, for example, the amount of drug accumulated in the patient's body and / or the specific efficacy of the delivery system of the present invention. Generally, the dosage can be calculated based on the EC50 determined to be effective in in vivo animal models and in vitro. For example, the dosage may be 0.01 μg to 1 g per kg of body weight. It can be administered daily, weekly, monthly, or yearly, once or several times per unit period, or it can be administered continuously over a long period using an infusion pump. The number of repeated administrations is determined based on factors such as the drug's retention time in the body and the drug concentration in the body. Even after treatment with a disease, the composition can be administered to treat recurrence.

[0036] The compositions of the present invention may further contain one or more active ingredients exhibiting the same or similar functions in relation to cancer treatment, or compounds that maintain / increase the solubility and / or absorption of the active ingredients, and may optionally further contain chemotherapeutic agents, anti-inflammatory agents, antiviral agents, and / or immunomodulatory agents.

[0037] The compositions of the present invention can be formulated by methods known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal, and the dosage form may be in the form of powder, granules, tablets, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution, or sterile powder.

[0038] All nucleic acid sequences described herein may be modified to a certain extent, and those skilled in the art will readily understand that nucleic acid sequences that maintain 70% or more homology through such artificial modifications are equivalent to those derived from the nucleic acid sequences of the present invention, as long as they retain the desired activity of the present invention.

[0039] The term "homology" refers to the degree of similarity with the presented nucleic acid sequence, and homology comparison can be performed visually or by calculating the percentage (%) of homology between two or more sequences using a readily available comparison program. The present invention includes nucleic acid sequences that are preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and most preferably 95% or more identical to the nucleic acid sequences presented in the present invention.

[0040] In the present invention, the term "plasmid" means a genetic construct containing essential regulatory elements such as a promoter so that a gene of interest can be expressed in a suitable host, and may be in a form integrated into the genome of a host cell or microorganism.

[0041] As used herein, "operably linked" refers to the functional linkage between a promoter or its variant nucleic acid sequence and a nucleic acid sequence encoding a target protein so that they can perform their general functions. Operable linkage with a plasmid can be achieved using recombinant DNA techniques well known in the art, and site-specific DNA cleavage and ligation can be achieved using enzymes generally known in the art.

[0042] In the present invention, the term "regulatory element" refers to a non-translated nucleic acid sequence that aids in or influences the transcription, translation, or expression of a nucleic acid sequence encoding a protein. The plasmid platform of the present invention may include a promoter or a variant thereof as a regulatory element, and may also include expression control sequences that can influence protein expression, such as an initiation codon, a stop codon, a polyadenylation signal, an enhancer, a signal sequence for membrane targeting or secretion, etc.

[0043] Furthermore, when the plasmid of the present invention is a replicable expression plasmid, it may contain a replication origin, which is a specific nucleic acid sequence from which replication is initiated.

[0044] The plasmid of the present invention may also contain a selection marker. The selection marker is used to select cells or microorganisms transformed with the plasmid, and a marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein, can be used. Only cells or microorganisms that express the selection marker survive in an environment treated with a selective agent, making it possible to select transformed individuals.

[0045] In the plasmid of the present invention, a gene encoding a target biomolecule may be operatively linked to the promoter, specifically, linked to a lower region of the promoter.

[0046] In one aspect of the present invention, a plasmid platform for stable transfer of nucleic acid molecules is provided, which includes a nucleic acid sequence encoding a modified protein from Lysosome-Associated Membrane Glycoprotein 2B (LAMP-2B) in which both the intracellular and extracellular domains have been deleted; a nucleic acid sequence encoding a protein that specifically binds to a nucleic acid molecule to be transferred; and a nucleic acid sequence encoding a glycosylated region.

[0047] The nucleic acid sequence encoding the modified protein may have the nucleic acid sequence of SEQ ID NO:4.

[0048] The nucleic acid sequence encoding the glycosylated region may have at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11 to 13, and from the viewpoint of simultaneously maximizing stable expression and transfer efficiency of the biomolecule, it may preferably have the nucleic acid sequence of SEQ ID NO: 11.

[0049] The nucleic acid sequence encoding the glycosylation region may be contained in the plasmid platform without any particular location limitations, provided that it can be contained in the plasmid platform. For example, it may be contained so that it is located in the direction of the extracellular region (or the outer region of the phospholipid bilayer) relative to the nucleic acid sequence encoding the modified protein. More specifically, a sequence encoding a biomolecule (or target protein) may be located in the direction of the extracellular region of the nucleic acid sequence encoding the modified protein, and the nucleic acid sequence encoding the glycosylation region may be located upstream (or toward the extracellular region) or downstream (or toward the intracellular region) of the sequence encoding the biomolecule, but is not particularly limited thereto. According to a preferred embodiment, the nucleic acid sequence encoding the glycosylation region may be located in the direction of the extracellular region relative to the nucleic acid sequence encoding the modified protein.

[0050] The nucleic acid sequence encoding a protein that specifically binds to the nucleic acid molecule is not limited to a particular location as long as it is contained in the plasmid platform and can be expressed so as to specifically bind to the nucleic acid molecule to be delivered, but for example, it can be contained so that it is located toward the intracellular region (or the inner region of the phospholipid bilayer) based on the nucleic acid sequence encoding the modified protein. More specifically, the sequence encoding the protein that specifically binds to the nucleic acid molecule can be located downstream (or toward the intracellular region) of the nucleic acid sequence encoding the modified protein, and the nucleic acid sequence encoding the glycosylation region can be located upstream (or toward the extracellular region) of the sequence encoding the modified protein, but is not particularly limited thereto.

[0051] The protein that specifically binds to the nucleic acid molecule may be at least one selected from the group consisting of a double-stranded ribonucleic acid binding motif, a binding protein derived from Bovine Immunodeficiency Virus (BIV), a binding protein derived from Jembrana Disease Virus (JDV), a binding protein derived from Human Immunodeficiency Virus (HIV), and mutants derived from the above proteins. According to a preferred embodiment, the protein may be a binding protein derived from JDV, but is not particularly limited thereto.

[0052] According to a specific embodiment, the nucleic acid sequence encoding a protein that specifically binds to the nucleic acid molecule may include a nucleic acid sequence having 70% or more homology to at least one sequence selected from the group consisting of SEQ ID NOs: 30 to 34, but is not particularly limited thereto.

[0053] The protein that specifically binds to the nucleic acid molecule may include a domain that binds to a specific motif contained in the nucleic acid molecule to be delivered, which may be, but is not limited to, a domain that specifically binds to a TAR sequence.

[0054] In one aspect of the present invention, there is provided a recombinant plasmid for stable transfer of a nucleic acid molecule, comprising the sequence of the nucleic acid molecule to be transferred within the aforementioned plasmid platform.

[0055] The position of the nucleic acid molecule within the plasmid is not particularly limited as long as the sequence of the nucleic acid molecule can be cleaved by an appropriate splicing process in the state of the plasmid injected into cells and can maintain stable binding to a protein that specifically binds to the nucleic acid molecule. In a preferred embodiment, the sequence of the nucleic acid molecule can be located between the nucleic acid sequence encoding the modified protein and the nucleic acid sequence encoding the protein that specifically binds to the nucleic acid molecule.

[0056] The nucleic acid molecule may be at least one selected from the group consisting of DNA, RNA and an aptamer, but is not limited thereto.

[0057] The nucleic acid molecule may contain a specific motif that enables it to maintain stable binding with a protein that specifically binds to the nucleic acid molecule. Specific examples include, but are not limited to, at least one selected from the group consisting of a TAR sequence, a modified TAR sequence, a short double-stranded DNA (preferably 7-27, 9-27, 11-27, 13-27, 7-25, 9-25, 11-25, 13-25, or 14-25 bp), and a short double-stranded RNA (preferably 7-27, 9-27, 11-27, 13-27, 7-25, 9-25, 11-25, 13-25, or 14-25 bp).

[0058] In one aspect of the present invention, there is provided an exosome for stable delivery of a nucleic acid molecule, comprising a product expressed from the recombinant plasmid described above.

[0059] In the exosome, the recombinant plasmid may further include a sequence encoding a protein that is expressed outside the exosome and specifically binds to the surface of a target cell, which may increase the targetability of delivery to the target cell and the delivery efficiency of the nucleic acid molecule to be delivered.

[0060] In the exosome, the protein that specifically binds to the nucleic acid molecule may be expressed inside the exosome and bind to the nucleic acid molecule, which may play a role in stabilizing the nucleic acid molecule inside the exosome to prevent degradation and enabling it to be stably delivered to the final target cell.

[0061] In one aspect of the present invention, there is provided a composition for stable delivery of a nucleic acid molecule comprising the exosome described above.

[0062] Furthermore, one aspect of the present invention provides a composition for RNA interference, which comprises an exosome, and the nucleic acid molecule has an RNA interference effect.

[0063] The nucleic acid molecule having the RNA interference effect may be, as a non-coding RNA, preferably at least one selected from the group consisting of miRNA, shRNA, siRNA, piRNA (piwi-interacting RNA), and lncRNA (long non-coding RNA), but is not limited thereto.

[0064] In one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising the exosome described above, wherein the recombinant plasmid further comprises a sequence encoding a protein that is expressed outside the exosome and specifically binds to the surface of a cancer cell, and the nucleic acid molecule has a preventive or therapeutic effect against cancer.

[0065] The composition comprises exosomes expressing in their outer regions a molecule that specifically binds to a protein specifically expressed on the surface of cancer cells, and the molecule expressed in the outer regions specifically binds to the surface of cancer cells, thereby enabling the exosomes to target cancer cells, making it possible to utilize the composition in cancer prevention or treatment applications based on such targeting.

[0066] Furthermore, the nucleic acid molecules contained in the exosomes may have a preventive or therapeutic effect against cancer, and such substances may be expressed in the inner bilayer region of the exosomes and then delivered to target cancer cells to exert their effects. In this case, the exosomes may deliver the nucleic acid molecules into cancer cells through interactions with cancer cells (e.g., surface receptor interaction, membrane fusion, receptor-mediated endocytosis, phagocytosis, micropinocytosis, etc.), thereby exerting a preventive or therapeutic effect against cancer.

[0067] The cancers include carcinomas, including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, and skin cancer, including squamous cell carcinoma; lymphoid hematopoietic malignancies, including leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, and Burkitt's lymphoma; myeloid hematopoietic malignancies, including acute and chronic myeloid leukemia and promyelocytic leukemia; fibrosarcoma and striated muscle other tumors including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, and according to a preferred embodiment, may be any one selected from the group consisting of, but not limited to, ovarian cancer.

[0068] It is obvious to a person skilled in the art that the detailed description of the invention related to the plasmid platform for stable transfer of the nucleic acid molecule can be interpreted with reference to the detailed description of the invention related to the plasmid platform for stable expression and transfer of the biomolecule. [Effects of the Invention]

[0069] The plasmid platform of the present invention contains a nucleic acid sequence encoding a modified protein of Lysosome-Associated Membrane Glycoprotein 2B (LAMP-2B) in which the intracellular domain, the extracellular domain, or a combination thereof has been deleted, enabling stable expression and delivery of biomolecules.

[0070] The recombinant plasmid of the present invention further comprises a sequence encoding a biomolecule to be expressed and transferred within the plasmid platform, thereby enabling stable expression and transfer of the biomolecule.

[0071] The exosomes of the present invention contain the recombinant plasmid and are capable of stable expression and delivery of biomolecules.

[0072] The composition for diagnosing cancer of the present invention comprises the exosome, and the biomolecule is a peptide that specifically binds to a protein specifically expressed on the surface of cancer cells, enabling effective diagnosis of cancer.

[0073] The pharmaceutical composition for preventing or treating cancer of the present invention comprises the exosome, and the biomolecule comprises a substance that specifically binds to a protein specifically expressed on the surface of cancer cells and a therapeutic substance to be delivered into cancer cells, thereby enabling effective prevention and treatment of cancer.

[0074] However, it should be understood that the effects are not limited to those described above, but include all effects that can be inferred from the configuration of the invention described in the detailed description or claims. [Brief explanation of the drawings]

[0075] [Figure 1] 1 to 5 are diagrams illustrating a plasmid platform proposed as one embodiment of the present invention. [Figure 2] Same as above. [Figure 3] Same as above. [Figure 4] Same as above. [Figure 5] Same as above. [Figure 6] FIG. 6 shows the results of Western blot analysis of the expression levels of target biomolecules for LAMP2-GFP, LAMP2-ΔIC-GFP, LAMP2-ΔEC-GFP, and LAMP2-ΔEC / IC-GFP. [Figure 7] FIG. 7 is a quantitative graph of the Western blot results of FIG. [Figure 8] FIG. 8 shows the results of microscopic observation of GFP fluorescence levels after overexpression of the recombinant plasmids used in the experiment in FIG. [Figure 9] FIG. 9 shows the results of microscopic observation of GFP fluorescence levels after overexpression of LEL-LAMP2-ΔEC-GFP and LEL-LAMP2-ΔEC / IC-GFP. [Figure 10] FIG. 10 shows the results of Western blotting to confirm the expression levels of target biomolecules for GNSTM-LAMP2-GFP, GNSTM-LAMP2-ΔIC-GFP, and GNSTM-LAMP2-ΔEC / IC-GFP. [Figure 11] FIG. 11 is a quantitative graph of the Western blot results of FIG. [Figure 12] FIG. 12 shows the results of microscopic observation of GFP fluorescence emission levels after overexpression of the recombinant plasmids used in the experiment of FIG. [Figure 13] FIG. 13 shows the results of microscopic observation of GFP fluorescence levels after overexpression of SEL-LAMP2-ΔEC-GFP and SEL-LAMP2-ΔEC / IC-GFP. [Figure 14] FIG. 14 shows the results of microscopic observation of GFP fluorescence levels after overexpression of GNSTM-LAMP2-ΔEC / IC-GFP and Gly-LAMP2-ΔEC / IC-GFP. [Figure 15] Figure 15 shows the results of Western blot analysis of the expression levels of target biomolecules for GNSTM-LAMP2-GFP, GNSTM-LAMP2-△IC-GFP, GNSTM-LAMP2-△EC / IC-GFP, GNSTM-LAMP2-EC5△IC-GFP, Gly-LAMP2-EC△IC-GFP, and LAMP2-EC25△IC-GFP. [Figure 16] FIG. 16 shows the results of microscopic observation of the specific binding ability of exosomes to cancer cells based on GFP fluorescence emission levels when a cancer cell-targeting peptide is contained as an active molecule. [Figure 17] Figures 17 to 19 show the results of examining the size of exosomes after overexpressing exosomes from cell lines not transformed with a plasmid (Figure 17), GNSTM-LAMP2-GFP (Figure 18), and GNSTM-LAMP2-△EC / IC-GFP (Figure 19). [Figure 18] Same as above. [Figure 19] Same as above. [Figure 20] Figure 20 shows the expression levels of GNSTM-LAMP2-△EC / IC-shGFP(w / o BIV), GNSTM-LAMP2-△EC / IC-shGFP-BIV(w / BIV), GNSTM-LAMP2-△EC / IC-shGFP-JDV(WT) (JDV_WT), and GNSTM-LAMP2-△EC / IC-shGFP-JDV(MT) (JDV_MT) in transfected cells and exosomes secreted from these cells. [Figure 21]Figure 21 shows the results of qPCR analysis of miRNA expression levels in a cell line not transfected with a plasmid (Control), a cell line transfected with no miRNA (SEL-LAMP2-△EC / IC-RBP with no miRNA) (w / o miRNA), and a cell line transfected with miRNA (SEL-LAMP2-△EC / IC-RBP with miRNA) (w / miRNA), as well as in target cells treated with exosomes secreted from these cells. [Figure 22] Figure 22 shows the level of GFP RNA expression suppression when GFP-expressing cells were treated with exosomes isolated from a cell line not transformed with a plasmid (Control) and cell lines transformed with the plasmids GNSTM-LAMP2-△EC / IC-scrambleGFP (scramble GFP), GNSTM-LAMP2-△EC / IC-shGFP-BIV (BIV shGFP), GNSTM-LAMP2-△EC / IC-shGFP-JDV(WT) (JDV WT shGFP), and GNSTM-LAMP2-△EC / IC-shGFP-JDV(MT) (JDV MT shGFP). [Figure 23] Figure 23 is a graph quantifying the level of GFP expression suppression after Western blotting when GFP-expressing cells were treated with exosomes isolated from a cell line not transformed with a plasmid (Control) and cell lines transformed with the plasmids GNSTM-LAMP2-△EC / IC-scrambleGFP (scramble GFP), GNSTM-LAMP2-△EC / IC-shGFP-BIV (BIV shGFP), GNSTM-LAMP2-△EC / IC-shGFP-JDV(WT) (JDV WT shGFP), and GNSTM-LAMP2-△EC / IC-shGFP-JDV(MT) (JDV MT shGFP). [Figure 24] FIG. 24 shows the Western blot results of the graph in FIG. [Figure 25]Figure 25 shows the results of microscopic observation of the level of GFP fluorescence expression suppression when GFP-expressing cells were treated with exosomes isolated from a cell line not transformed with a plasmid (Control) and cell lines transformed with the plasmids GNSTM-LAMP2-△EC / IC-scrambleGFP (scramble GFP), GNSTM-LAMP2-△EC / IC-shGFP-BIV (BIV shGFP), GNSTM-LAMP2-△EC / IC-shGFP-JDV(WT) (JDV WT shGFP), and GNSTM-LAMP2-△EC / IC-shGFP-JDV(MT) (JDV MT shGFP). DETAILED DESCRIPTION OF THE INVENTION

[0076] Hereinafter, for more specific explanation, a detailed description will be given based on examples and experimental examples. However, the following examples and experimental examples are merely illustrative and do not limit the scope of the present invention. [Example]

[0077] Example 1. Preparation of recombinant plasmids containing genes encoding modified peptides The entire sequence encoding LAMP-2B (NM_013995.2) was synthesized and amplified by PCR. The amplified sequence was then inserted into the pcDNA 3.1(+) vector (or into an animal cell overexpression vector) using an Infusion cloning kit (In-Fusion® HD cloning kit, clontech, Cat. No. 639648) to prepare a plasmid containing LAMP2B. In this plasmid, the sequence encoding HA (Hyaluronic Acid), the sequence encoding the target peptide, and the sequence encoding the active protein were synthesized and amplified by PCR, and inserted into each sequence position using an infusion cloning kit to create a recombinant plasmid (SP: Signal Peptide, sequence encoding the signal peptide; HA: Hyaluronic Acid, sequence encoding hyaluronic acid; Target Peptide: sequence encoding the target peptide, sequence encoding the FLAG protein; Active Protein: sequence encoding the active protein, sequence encoding GFP). In addition, a mutagenesis kit (EZchange) was used in addition to the above method. TM Using the Site-directed Mutagenesis kit (Enzynomics, Cat. No. EZ004S), we prepared a recombinant plasmid (LAMP2-△IC-GFP) in which the sequence encoding the intracellular domain (IC) of LAMP-2B was deleted, a recombinant plasmid (LAMP2-△EC-GFP) in which the sequence encoding the extracellular domain (EC) of LAMP-2B was deleted, and a recombinant plasmid (LAMP2-△EC / IC-GFP) in which the sequence encoding both the intracellular and extracellular domains of LAMP-2B was deleted. These plasmids are shown in Figure 1.

[0078] In addition, for a part of the backbone structure in Figure 1, the sequence encoding the extracellular domain (EC) region of LAMP-2B was synthesized and PCR-amplified with the sequence encoding the CD9 LEL (Large Extracellular Domain of CD9) in tetraspanin, and replaced with the sequence using the infusion cloning kit method to produce recombinant plasmids (LEL-LAMP-△EC-GFP, LEL-LAMP-△EC / IC-GFP), which are diagrammed as shown in Figure 2.

[0079] In addition, for a part of the backbone structure in Figure 1, in order to introduce glycosylation in the upstream (towards the outside of the cell) or downstream (towards the inside of the cell) region of the target peptide, (1) a sequence encoding a GNSTM motif (GNSTM is an amino acid sequence that is most strongly N-glycosylated in cells) was inserted into the relevant position using a mutagenesis kit (EZchange TM (2) The sequence encoding the tetraspanin CD9 SEL (CD9 small extracellular domain) was synthesized and amplified by PCR and then introduced into the conventional LAMP2B-GFP modified plasmid using an infusion cloning kit (SEL-LAMP2-△EC-GFP, SEL-LAMP2-△EC / IC-GFP). (3) The N-glycosylation sequence AAC was introduced downstream of the sequence encoding the FLAG protein of LAMP2-βEC / IC-GFP using a mutagenesis kit (EZchange™ Site-directed Mutagenesis Kit, Enzynomics, Cat. No. EZ004S) to prepare recombinant plasmids, which are shown in Figure 3.

[0080] In addition, a part of the backbone structure in Figure 3 (EC25) of the sequence encoding the extracellular domain of LAMP-2B was mutagenized using a mutagenesis kit (EZchange TM The recombinant plasmids (LAMP2-EC25△IC-GFP, GNSTM-LAMP2-EC25△IC-GFP, Gly-LAMP2-EC25△IC-GFP) were prepared using the Site-directed Mutagenesis kit (Enzynomics, Cat No. EZ004S), and are shown in Figure 4.

[0081] In addition, for a portion of the backbone structure shown in Figure 3, the sequence encoding the active protein in GNSTM-LAMP-GFP-ΔEC / IC or SEL-LAMP2-ΔEC / IC was replaced with a sequence encoding a protein that binds to (sh)RNA. Pri-miRNA sequences were synthesized between the HA-encoding sequence and the (sh)RNA-binding protein (RBP)-encoding sequence and amplified by PCR. Recombinant plasmids (GNSTM-LAMP2-ΔEC / IC-RBP, SEL-LAMP2-ΔEC / IC-RBP) were then prepared using an infusion kit, as shown in Figure 5. The Pri-miRNA used consisted of the Pri-miRNA base sequence of miRNA-199 and the Pri-miRNA sequence of shGFP, which contained a GFP target sequence in the miRNA-199 base sequence. The Pri-miRNA sequence contained a TAR sequence, and the RBP was designed to incorporate a protein that specifically binds to the TAR site. Therefore, we used wild-type protein derived from BIV (Bovine Immunodeficiency Virus) and wild-type (WT) and mutant (MT) proteins derived from JDV (Jembrana Disease Virus) as the (sh)RNA-binding proteins (RNA binding proteins; RBPs).

[0082] The specific sequences of the recombinant plasmids prepared above are shown in Table 1 below.

[0083] Table 1-1-1 Table 1-1-2 Table 1-1-3

[0084] Table 1-2-1 Table 1-2-2 Table 1-2-3

[0085] Table 1-3-1 Table 1-3-2 Table 1-3-3

[0086] Table 1-4

[0087] Table 1-5

[0088] Example 2. Expression of recombinant plasmids To express the recombinant plasmid, 2.5 x 10 HEK293T cells 6 The number of cells was 100 mm 2 Culture dishes were filled with 10% Fetal Bovine Serum (FBS) (Gibco TM , Cat No. 16000044) and 1% antibiotic (1% Penicillin / Streptomycin, Gibco TM The cells were cultured for 24 hours in DMEM (Welgene, Cat. No. LM001-05) containing PEG-1000 (Cat. No. 15140122). The recombinant plasmid pcDNA3.1(+) vector containing the target gene was then transferred to the PolyJet TM 2 μg of the antibody was introduced into the cells using a transfection kit (Signagen® Laboratories, Cat No. SL100688) and the cells were cultured for 48 hours.

[0089] Example 2-1. Fluorescence imaging experiment To confirm intracellular expression of recombinant plasmid fluorescence, the medium was replaced with DMEM without fetal bovine serum and cultured for an additional 48 hours. After the culture was completed, intracellular green fluorescence expression was confirmed using Cytation 5 (Biotek).

[0090] Example 2-2. Exosome isolation, protein expression, RNA expression, etc. After washing once with 1X DPBS (Welgene, Cat. No. LB001-02), 1 mL of trypsin-EDTA (Welgene, Cat. No. LS015-10) was added to the culture dish to detach the cells. Then, 10 mL of DMEM medium containing fetal bovine serum and antibiotics was added, and the cells were collected and centrifuged at 1000 rpm for 2 minutes. The medium was removed, and the collected cells were dissociated by adding 10 mL of DMEM medium without fetal bovine serum. After that, the cells were transferred to a suspension culture dish (SPL Life Science, Cat. No. 11151) and further cultured in DMEM medium containing only 1% antibiotics for 48 hours. After that, exosomes and cells were isolated and used for protein and RNA expression experiments.

[0091] Experimental Example 1. Stable expression and target delivery of active proteins using modified peptides 1. Confirmation of the effect of increasing the expression level of the target protein after removal of the intracellular domain (IC) and extracellular domain (EC). (1) Experimental method A gene encoding a FLAG protein as a targeting peptide and a gene encoding a GFP as an active protein were inserted into the plasmid backbone structures of Figures 1 and 2, respectively, and then each recombinant plasmid was overexpressed by the method of Example 2-1 or 2-2.

[0092] That is, to confirm the actual fluorescence expression of the cells, each recombinant plasmid was overexpressed according to the method of Example 2-1, and then the fluorescence expression was measured.

[0093] To confirm protein expression levels, after culturing as described in Example 2-2, cells and culture medium were collected into a 50 mL conical tube (SPL, Cat. No. 50040) and centrifuged at 1000 rpm for 2 minutes. The collected cells were washed twice with 1X PBS and then diluted with 1X cell lysis buffer (10X cell lysis buffer, Cell Signaling Technology, Cat. No. 9803) supplemented with PMSF to isolate proteins from the cells. The isolated culture medium was then centrifuged again at 4000 rpm for 30 minutes, after which cellular debris was removed. The supernatant was then placed into a 10 kDa filter tube (Millipore, Cat. No. UFC9010) and centrifuged at 4000 rpm for 30 minutes. The final supernatant was mixed with 1X PBS and centrifuged twice. This process was repeated twice, and the supernatant remaining in the filter tube was filtered through a 0.2μm filter using a 27G needle syringe to obtain single-sized exosomes. The expression levels of recombinant proteins in the cells and exosomes were compared using Western blot analysis of the cell lysate and the obtained exosomes.

[0094] (2) Experimental results When overexpressing the plasmids shown in Figure 1, the expression levels of FLAG protein and GFP were significantly higher when recombinant plasmids containing sequences encoding the intracellular domain (LAMP2-△IC-GFP), the extracellular domain (LAMP2-△EC-GFP), or all of these (LAMP2-△EC / IC-GFP) were overexpressed compared to when the entire LAMP-2B coding sequence was overexpressed (Figures 6 and 7). In particular, in the case of LAMP2-△EC / IC-GFP, FLAG protein levels increased approximately 6- to 20-fold in cells, and GFP protein levels increased approximately 50- to 100-fold in cells and exosomes compared to LAMP2-GFP (Figures 6A-B). In fact, the fluorescence measurement results confirmed that the fluorescence intensity of LAMP2-△IC-GFP, LAMP2-△EC-GFP, and LAMP2-△EC / IC-GFP was significantly stronger than that of LAMP2-GFP, and that the fluorescence intensity of LAMP2-△EC / IC-GFP was particularly strong (Figure 8).

[0095] This demonstrates that deleting the intracellular domain of LAMP2 can increase the stable expression of both the target protein and active protein in exosome-secreting cells or exosomes. Furthermore, deleting the extracellular domain of LAMP2 significantly increases expression stability. LAMP2 is known to be involved in lysosomal degradation, and deleting its intracellular domain is expected to avoid lysosomal degradation and increase the expression rate of the target protein. However, deleting the extracellular domain of LAMP2 significantly reduced the stability of LAMP2 itself, which would have been expected to have a negative effect on the expression rate of the target protein. However, the significant increase in expression rate was an unexpected effect, and further research is needed.

[0096] Furthermore, when the plasmid in Figure 2 was overexpressed, it was confirmed that the fluorescence intensity was significantly stronger when the sequence encoding the intracellular domain of LAMP2 was removed (LEL-LAMP2-△EC / IC) than when it was not (LEL-LAMP2-△EC) (Figure 9).

[0097] This more clearly demonstrates that removing the intracellular domain of LAMP2 can increase the expression levels of target proteins and active proteins by suppressing the lysosomal degradation of cells caused by the influx of extracellular and extracellular proteins via the intracellular domain. The same results were obtained when the extracellular domain region of LAMP2 was replaced with the extracellular domain region of other proteins.

[0098] 2. Confirmation of the effect of glycosylation on increasing expression levels (1) Experimental method A gene encoding the FLAG protein as the targeting peptide and a gene encoding GFP as the active protein were inserted into the plasmid backbone structures shown in Figures 3 and 4, respectively, and then the respective recombinant plasmids were overexpressed by the methods of Examples 2-1 and 2-2. The other experimental methods were as described in Experimental Example 1-1-(1) above.

[0099] (2) Experimental results When the plasmids shown in Figure 3 were overexpressed, the plasmid containing the entire LAMP2 sequence in the GNSTM motif sequence (GNSTM-LAMP2-GFP) showed very low expression levels of the target protein, FLAG, and the active protein, GFP (Figure 10). This result is inconsistent with previous reports, suggesting that the GNSTM motif does not actually stabilize the target protein in LAMP2. However, when the intracellular domain of LAMP2 was deleted (GNSTM-LAMP2-△IC-GFP) or when the extracellular domain was also deleted (GNSTM-LAMP2-△EC / IC-GFP), the GNSTM motif significantly stabilized the expression of the target protein. In particular, when both the intracellular and extracellular domains of LAMP2 were completely removed (GNSTM-LAMP2-△EC / IC-GFP), the expression levels of both the target protein, FLAG, and the active protein, GFP, were significantly higher. Compared to when the GNSTM motif sequence was combined with the entire LAMP2 sequence, FLAG expression increased 200-900-fold, and GFP expression increased 40-80-fold (Figure 11). The fluorescence levels also reflected these results, with GNSTM-LAMP2-△IC-GFP showing higher fluorescence intensity than GNSTM-LAMP2-GFP, and GNSTM-LAMP2-△EC / IC-GFP showing a marked difference in intensity (Figure 12).

[0100] This tendency was also observed when the target protein contained the CD9 SEL domain, which is known to contain glycosylated amino acids, in a subregion. However, when the intracellular domain of LAMP2 was further removed (SEL-LAMP2-△EC / IC-GFP) compared to when the extracellular domain of LAMP2 was replaced with SEL (SEL-LAMP2-△EC-GFP) the fluorescence intensity was even stronger (Figure 13).

[0101] In addition, we used a mutagenesis kit to replace the glycosylation-active amino acid GNG (Glycine-Asparagine-Glycine) at the middle site of the linker located downstream of FLAG in the LAMP2-ΔEC / IC-GFP plasmid. Specifically, the linker amino acid sequence GSSGGGSG (DNA sequence: ggctcgagtGgtggaggatctggt) was modified with GSSGNGSG (DNA sequence: ggctcgagtGgtAACggatctggt). The same trend was observed when glycosylation was performed at a lower region of the target protein (Gly-LAMP2-ΔEC / IC-GFP). When both the extracellular and intracellular domains of LAMP2 were completely deleted, the fluorescence intensity was significantly enhanced, similar to that observed with the introduction of the GNSTM motif and the introduction of SEL (Figure 14).

[0102] Furthermore, we anticipated that including a portion of the extracellular domain of LAMP2 would further enhance target protein stabilization depending on the location and structure of glycosylation. We constructed plasmids (LAMP2-EC25△IC-GFP, GNSTM-LAMP2-EC25△IC-GFP, and Gly-LAMP2-EC25△IC-GFP) containing nucleic acid sequences encoding the last 25 amino acids of LAMP2, and then examined the expression levels of the target protein. Compared to GNSTM-LAMP2-GFP, GNSTM-LAMP2-EC25△IC-GFP, Gly-LAMP2-EC25△IC-GFP, and GNSTM-LAMP2-△EC / IC-GFP all showed higher expression levels of the target protein FLAG and the active protein GFP, with GNSTM-LAMP2-△EC / IC-GFP being particularly high. This suggests that the highest target protein expression was achieved when both the extracellular and intracellular domains of LAMP2 were completely removed. Furthermore, LAMP2-EC25△IC-GFP showed even higher expression levels of the target protein, which is thought to indicate that the GNSTM motif does not exert an expression stabilizing effect in the presence of the extracellular domain of LAMP2 (Figure 15).

[0103] This indicates that for the previously known GNSTM motif to act together with LAMP2 and exhibit the effect of stabilizing the expression of a target protein, at least the intracellular domain of LAMP2 must be deleted; when the extracellular domain is also deleted, the expression level of the target protein can be significantly increased, a result that could not easily be predicted from what was previously known.

[0104] 3. Targeting cancer cells using exosomes (1) Experimental method In the plasmid backbone structure shown in Figure 3, we confirmed that the expression rate of GNSTM-LAMP2-△EC / IC-GFP FLAG in cells and exosomes was significantly increased when a plasmid containing GNSTM was used. The target peptide site of this plasmid was replaced with a nucleic acid sequence encoding a cancer targeting peptide sequence ("MHTAPGWGYNLS") (folate receptor binding peptide) (Table 2 below). Each recombinant plasmid was then overexpressed as described in Example 2-2. After incubation, the cells and culture medium were collected in a 50 mL conical tube (SPL, Cat. No. 50040) and centrifuged at 1000 rpm for 2 minutes. The supernatant was then centrifuged at 4000 rpm for 30 minutes to remove cellular debris. The final supernatant was placed in a 10 kDa filter tube (Millipore, Cat. No. UFC9010) and centrifuged at 4000 rpm for 30 minutes. The final supernatant was mixed with 1X PBS and centrifuged. This process was repeated twice. The supernatant remaining in the filter tube was then filtered through a 0.2 μm filter using a syringe with a 27-gauge needle to obtain mono-sized exosomes. The exosomes were quantified using a BCA kit, and 100 μg of exosomes were stained using the PKH67 Green Fluorescent Fine Linker Kit (Sigma, Cat No. PKH67GL). The stained exosomes were placed on a 100 kDa filter (Millipore, Cat No. UFC510008) and centrifuged at 14,000 rcm for 5 minutes. The supernatant was then added to 1X PBS and centrifuged again. This process was repeated twice. The final exosome product was filtered through a 0.2 μm filter with a 27-gauge needle and then applied to SKOV3 cells, a folate receptor-expressing ovarian cancer cell line, and cultured for 24 hours. After incubation, the cells were washed twice with 1X DPBS and then replaced with SKOV3 culture medium, and the level of green fluorescence was measured using Cytation 5 (Biotek).

[0105] [Table 2]

[0106] (2) Experimental results The results show a comparison of the fluorescence expression in SKOV3 cells treated with exosomes that do not overexpress the plasmid (exosomes (+)), exosomes with the FLAG sequence of GNSTM-LAMP2-△EC / IC-GFP inserted (control peptide exosomes (+)) as shown in Figure 3, and exosomes with a cancer-targeting peptide inserted at the FLAG position of GNSTM-LAMP2-△EC / IC-GFP (targeting peptide exosomes (+)). The fluorescence expression of the overexpressing exosomes was significantly stronger and distributed in greater quantities than the other two groups (Figure 16).

[0107] This confirmed that the plasmid carrying the sequence encoding the target peptide was stably expressed in cells and exosomes, exhibited activity on the surface of exosomes, and could be selectively transported in large amounts to cells expressing the cancer target peptide-binding receptor, suggesting its potential use as a therapeutic agent for cancer targeting through the effective expression of biomolecules.

[0108] 4. Exosome Size Distribution Measurement (1) Experimental method Using the method described in Example 2-2, exosomes (Figure 17), GNSTM-LAMP2-GFP (Figure 18), and GNSTM-LAMP2-△EC / IC-GFP (Figure 19) were overexpressed in cell lines not transformed with a plasmid. After incubation, the cells and culture medium were collected in a 50 mL conical tube (SPL, Cat. No. 50040) and centrifuged at 1,000 rpm for 2 minutes. The supernatant was then centrifuged at 4,000 rpm for 30 minutes to remove cellular debris. The final supernatant was placed in a 10 kDa filter tube (Millipore, Cat. No. UFC9010) and centrifuged at 4,000 rpm for 30 minutes. The final supernatant was mixed with 1X PBS and centrifuged again. This process was repeated twice, and the supernatant remaining in the filter tube was filtered through a 0.2 μm filter using a 27-G needle and syringe to obtain mono-sized exosomes. The size of the harvested exosomes was measured by DLS (Dynamic Light Scattering).

[0109] (2) Experimental results As can be seen from Figures 17 to 19, there was no difference in size between exosomes from cell lines not transformed with a plasmid (Figure 17), exosomes overexpressing GNSTM-LAMP2-GFP (Figure 18), and exosomes overexpressing GNSTM-LAMP2-△EC / IC-GFP (Figure 19). Despite the removal of both the extracellular and intracellular domains of LAMP2, there was no difference in exosome size upon overexpression. This suggests that the use of LAMP2-△EC / IC of the present invention does not alter the basic properties of exosomes after overexpression, suggesting that there are no problems with the use of conventional exosomes in various biological applications.

[0110] Experimental Example 2. Stable expression and target delivery of active RNA using modified peptides 1.Confirmation of expression level (1) Experimental method A gene encoding a FLAG protein as a targeting peptide and a gene encoding a protein that binds to RNA as an active protein were inserted into the plasmid backbone structure of Figure 5, and then each recombinant plasmid was overexpressed by the method of Example 2-2.

[0111] To confirm the active shRNA expression level in the cells, after the culture was completed according to the method of Example 2-2, the cells and culture medium were collected in a 50 mL conical tube (SPL, Cat. No. 50040) and centrifuged at 1000 rpm for 2 minutes. The collected cells were washed twice with 1X PBS and RNA was isolated using the Trizol method (ThermoFisher Scientific, Cat. No. 15596026). 500 ng of RNA was then polyadenylated using a polyadenylation kit (Enzynomics, Cat. No. EX041S) and purified using PrimeScript. TMcDNA was synthesized by the gene-specific primer cDNA synthesis method using a primer specific to the shRNA sequence introduced in the cDNA kit (Takara, Cat. No. 6210A). After cDNA synthesis, the target shRNA sequence was probed using the TaqMan probe for TOPreal TM The expression level of shRNA was measured using Probe qPCR PreMix (Enzynomics, Cat. No. RT600S).

[0112] To confirm the expression level of active shRNA in exosomes, after culturing as described in Example 2-2, cells and culture medium were collected in a 50 mL conical tube (SPL, Cat. No. 50040) and centrifuged at 1000 rpm for 2 minutes. The supernatant was then centrifuged at 4000 rpm for 30 minutes to remove cellular debris. The final supernatant was placed in a 10 kDa filter tube (Millipore, Cat. No. UFC9010) and centrifuged at 4000 rpm for 30 minutes. The final supernatant was mixed with 1X PBS and centrifuged again. This process was repeated twice, and the supernatant remaining in the filter tube was filtered through a 0.2 μm filter using a 27-G needle and syringe to obtain mono-sized exosomes. The collected exosomes were subjected to RNA isolation using the Trizol method (ThermoFisher Scientific, Cat. No. 15596026), and 500 ng of RNA was subjected to polyadenylation using a polyadenylation kit (Enzynomics, Cat. No. EX041S), followed by PrimeScript PCR. TM cDNA was synthesized by the gene-specific primer cDNA synthesis method using a primer specific to the shRNA sequence introduced in the cDNA kit (Takara, Cat. No. 6210A). After cDNA synthesis, the target shRNA sequence was probed using the TaqMan probe for TOPreal TM The expression level of shRNA was measured using Probe qPCR PreMix (Enzynomics, Cat. No. RT600S).

[0113] To measure GFP expression levels in SKOV3 cells treated with exosomes containing active shRNA, the cells and culture medium were collected in a 50 mL conical tube (SPL, Cat. No. 50040) and centrifuged at 1000 rpm for 2 minutes. The supernatant was then centrifuged at 4000 rpm for 30 minutes to remove cellular debris. The final supernatant was placed in a 10 kDa filter tube (Millipore, Cat. No. UFC9010) and centrifuged at 4000 rpm for 30 minutes. The final supernatant was mixed with 1X PBS and centrifuged again. This process was repeated twice, and the supernatant remaining in the filter tube was filtered through a 0.2 μm filter using a 27-G needle and syringe to obtain mono-sized exosomes.

[0114] To verify the efficacy of shGFP-containing exosomes in silencing GFP-overexpressing cells, SKOV3 cell line (1 × 10) was used. 5 The cells were then aliquoted into each well of a 6-well plate and cultured for 24 hours. TM 500 ng of the GFP fluorescent vector pAcGFP1-N1 (Clonetech, PT3716) was introduced into each well using a transfection kit (Signagen® Laboratories, Cat No. SL100688). After 24 hours, the culture medium was replaced with fresh medium. 48 hours after treatment, 50 μg of the isolated shRNA-containing exosomes were added to each well, and GFP fluorescence and protein expression levels were measured.

[0115] (2) Experimental results All four plasmid overexpression models (GNSTM-LAMP2-ΔEC / IC-shGFP, GNSTM-LAMP2-ΔEC / IC-shGFP-BIV, GNSTM-LAMP2-ΔEC / IC-shGFP-JDV(WT), and GNSTM-LAMP2-ΔEC / IC-shGFP-JDV(MT)) showed higher expression levels in cells than the exosome-only (NC) control group. In particular, when BIV-derived RBP and JDV-derived RBP were inserted into the active protein, the expression of shGFP in the whole exosome was significantly higher than in the uninserted (w / o BIV:RBP-less construct) group. Insertion of JDV wild-type RBP was the most effective, with an approximately 80-fold increase compared to insertion of BIV-derived RBP (Figure 20).

[0116] Furthermore, when the SEL-LAMP2-△EC / IC-(Pri-miRNA-199)-RBP plasmid, a glycosylation model using non-GNS™ SEL, was overexpressed, miRNA-199 was also well expressed in transfected cells (Figure 21).

[0117] These experimental results indicate that a form of LAMP2 in which both the extracellular and intracellular domains have been removed and glycosylation has been introduced can stably express RNA and active protein in both transfected cells and the resulting exosomes, ultimately suggesting the possibility of delivering bioactive nucleic acid molecules via exosomes.

[0118] 2. Check the transmission level When the SEL-LAMP2-△EC / IC-(Pri-miRNA-199)-RBP plasmid, a glycosylation model using SEL, was overexpressed, we confirmed that miRNA-199 was well expressed in cells treated with exosomes isolated after removing the transfected cells (Figure 21).

[0119] Furthermore, when four GNSTM-based glycosylation models, GNSTM-LAMP2-△EC / IC-shGFP-BIV, GNSTM-LAMP2-△EC / IC-shGFP-JDV(WT), and GNSTM-LAMP2-△EC / IC-shGFP-JDV(MT), were overexpressed, we confirmed that shGFP was well expressed in GFP-transfected SKOV3 cells treated with exosomes isolated after removal of transfected cells, and that shGFP suppressed GFP expression (at both RNA and protein levels) very effectively (Figures 22 and 23). In particular, the most effective RBP was observed when JDV(WT)-derived protein was used as the activated protein. This trend was also confirmed by Western blot and fluorescence analysis in target cells (Figures 24 and 25). Fluorescence analysis showed that overexpression of GNSTM-LAMP2-△EC / IC-shGFP-JDV(WT) plasmid significantly suppressed GFP mRNA and protein expression by 90-99%.

[0120] These experimental results indicate that the deletion of the extracellular and intracellular domains of LAMP2 and the introduction of glycosylation resulted in stable expression of RNA and active protein in transfected cells and exosomes, and ultimately resulted in efficient dissociation of the RNA in exosomes from RBP, resulting in successful delivery of RNA to target cells. In particular, the activity of shRNA was maximized when JDV-derived RBP and glycosylated LAMP2-△EC / IC were inserted into the plasmid.

[0121] The scope of the present invention is defined by the following claims, and it should be understood that all modifications and variations derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention.

Claims

1. A nucleic acid sequence encoding a modified protein in which the intracellular domain and extracellular domain of LAMP-2B (Lysosome-Associated Membrane Glycoprotein 2B) have been deleted; and a nucleic acid sequence encoding a biomolecule for expression and delivery; Including, The nucleic acid sequence encoding the modified protein has a nucleic acid sequence having 90% or more sequence identity with the nucleic acid sequence of SEQ ID NO:

4. Recombinant plasmids for stable expression and transfer of biomolecules.

2. The recombinant plasmid of claim 1 , wherein the recombinant plasmid further comprises a nucleic acid sequence encoding a glycosylated region.

3. The recombinant plasmid of claim 2 , wherein the nucleic acid sequence encoding the glycosylated region is located toward the extracellular region relative to the nucleic acid sequence encoding the modified protein.

4. The recombinant plasmid of claim 2, wherein the nucleic acid sequence encoding the glycosylated region comprises a nucleic acid sequence having 90% or more sequence identity with any one of the nucleic acid sequences of SEQ ID NOs: 11 to 13.

5. 3. The recombinant plasmid of claim 2, wherein the nucleic acid sequence encoding the glycosylated region comprises a nucleic acid sequence having 90% or more sequence identity with the nucleic acid sequence of SEQ ID NO:

11.

6. 2. The recombinant plasmid of claim 1, wherein the recombinant plasmid comprises a nucleic acid sequence having 90% or more sequence identity to the nucleic acid sequence of SEQ ID NO:4 and a nucleic acid sequence having 90% or more sequence identity to the nucleic acid sequence of SEQ ID NO:

11.

7. The recombinant plasmid of claim 1, wherein the nucleic acid sequence encoding the biological molecule is located i) between the nucleic acid sequence encoding a glycosylated region further contained in the recombinant plasmid and the nucleic acid sequence encoding the modified protein, ii) toward the intracellular region relative to the nucleic acid sequence encoding the modified protein, or iii) all of these.

8. 2. The recombinant plasmid according to claim 1, wherein the biomolecule is at least one selected from the group consisting of nucleic acid molecules, aptamers, peptides, proteins, glycoproteins, lipoproteins, immunoglobulins, hormones, growth factors, recombinases, and fluorescent proteins.

9. An exosome for stable expression and delivery of a biomolecule, comprising a product expressed from the recombinant plasmid of any one of claims 1 to 8.

10. The exosome of claim 9 , wherein the biological molecule comprises a substance that is expressed on the outside of the exosome and specifically binds to the surface of a target cell.

11. A composition for diagnosing cancer, comprising the exosome according to claim 9, wherein the biomolecule comprises one that specifically binds to a protein specifically expressed on the surface of cancer cells.

12. The composition of claim 11 , wherein the cancer is ovarian cancer.

13. A pharmaceutical composition for preventing or treating cancer, comprising the exosome of claim 9, wherein the biomolecule specifically binds to a protein specifically expressed on the surface of cancer cells, and a therapeutic substance to be delivered into cancer cells.

14. The pharmaceutical composition of claim 13 , wherein the cancer is ovarian cancer.

Images