Novel polypeptide compositions for intracellular transfection

Abstract

The present invention relates to a novel peptide composition for intracellular transfection and its use. The novel peptide composition for intracellular transfection according to the present invention has the advantages of significantly improving the transfection efficacy of target substances and significantly reducing cytotoxicity. As a result, it has various useful values ​​in terms of being able to deliver various substances into cells.

Description

[Technical Field]

[0001] The present invention relates to novel polypeptides for intracellular transfection and their uses. [Background technology]

[0002] Nucleic acid carriers for delivering nucleic acid substances into cells can be broadly divided into viral vectors and non-viral vectors.

[0003] Non-viral vectors can be used in a variety of formulations, including liposomes, cationic polymers, micelles, emulsions, and nanoparticles. In these formulations, cationic lipids are the core material in the design of nucleic acid delivery systems (lipofection) because they provide electrostatic binding to anionic nucleic acid substances. Cationic lipids form stable ionic complex particles with anionic nucleic acid substances, and these complexes are transported into cells via cell membrane fusion or endocytosis.

[0004] Previously developed cationic lipids were made cationic by combining a neutral fatty acid chain with an amine-containing compound such as a primary amine, secondary amine, tertiary amine, or quaternary ammonium salt.

[0005] Although these lipids have a relatively high gene transfer efficiency, they have been reported to be cytotoxic. To overcome this cytotoxicity, lipids using amino acid linkers instead of non-amino acid linkers have been synthesized.

[0006] Recent reports have shown that cationic lipids prepared by combining fatty acid amines with the carboxyl groups of amino acids are, contrary to expectations, often cytotoxic. In particular, most of the prepared cationic lipids have been reported to have very poor intracellular delivery efficiency of target substances such as oligonucleotides, rendering them of no practical value. This suggests that simply constructing a lipid delivery system by combining only amino acids with fatty acid amines is difficult to achieve intracellular delivery efficiency, and that delivery efficiency is determined by its specific structure. Therefore, it suggests that a practical delivery system can only be used after meticulous prior design and experimental results.

[0007] Although viral vectors have high gene transfer efficiency, they are pathogenic viruses, which pose safety issues and limit the size of the gene that can be inserted into the vector. Furthermore, numerous issues regarding their immunogenicity have recently emerged, which severely limits the use of viral vectors for nucleic acid transfer.

[0008] Under these circumstances, the present inventors have produced a novel nucleic acid transfer system and completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0009] One object of the present invention is to provide novel polypeptides for intracellular transfection.

[0010] Another object of the present invention is to provide a novel use of polypeptides for intracellular transfection. [Means for solving the problem]

[0011] This will be explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of various elements disclosed in the present invention belong to the scope of the present invention. Furthermore, it cannot be said that the scope of the present invention is limited by the following specific description.

[0012] Furthermore, the terms used in this specification are merely used for the purpose of description and should not be construed as limiting. The singular expressions include the plural expressions unless the context clearly indicates otherwise. In this specification, the terms "comprise" or "have" and the like specify the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should be understood not to preclude the presence or additional possibility of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0013] Furthermore, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and should not be interpreted in an ideal or overly formal sense unless expressly defined in this application.

[0014] In addition, in order to avoid confusion, the following description omits overlapping content. In other words, the content of the invention is not limited to the following content alone, and the content of the invention must be interpreted based on the overall content of the invention.

[0015] Polypeptides To achieve the above object, one aspect of the present invention provides a polypeptide comprising 9, 10, or 11 consecutive leucines linked to 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1.

[0016] More specifically, the present invention provides a polypeptide that is necessarily composed of 9, 10, or 11 consecutive leucines linked to 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1.

[0017] More specifically, the present invention provides a polypeptide consisting of 9, 10, or 11 consecutive leucines linked to 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1.

[0018] In the present invention, the term "peptide" refers to a molecule formed by amino acid residues linked to each other via amide bonds (or peptide bonds). The peptide may be synthesized using a gene recombination and protein expression system, or preferably, may be synthesized in vitro using a peptide synthesizer.

[0019] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (or peptide bonds). The term "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, and the like, all of which are used to refer to chains composed of two or more amino acids.

[0020] In the present invention, the terms "amino acid" and "amino acid residue" refer to natural amino acids, unnatural amino acids, and modified amino acids. Unless otherwise specified, all references to amino acids include references to both D and L stereoisomers (if the structure allows for such stereoisomeric forms), either generally or specifically according to the name. Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Unnatural amino acids include modified amino acid residues that have been chemically modified or chemically blocked, either reversibly or irreversibly, on the N-terminal amino group or side chain functional group, e.g., N-methylated D and L amino acids or residues in which the side chain functional group has been further chemically modified to another functional group.

[0021] A polypeptide according to the invention is a single polypeptide chain comprising fused components, which may be linked directly or indirectly.

[0022] The polypeptide according to the present invention may optionally include derivatives thereof, in which a fragment or a portion of the amino acids of the peptide is substituted or deleted, a portion of the amino acid sequence is modified to have a structure that increases stability in vivo, a portion of the amino acid sequence is modified to increase hydrophilicity, or some or all of the amino acids are substituted with L- or D-amino acids, or a fragment in which some of the amino acids are modified.

[0023] Specifically, the peptides according to the present invention may be modified at the N- and / or C-terminal amino acids of the polypeptide to increase the stability and biological activity of the peptide, for example, by N-terminal acetylation or C-terminal amidation.

[0024] According to the present invention, the term "linked thereto" refers to the linkage and / or binding of a peptide sequence to the N-terminus or C-terminus. Here, linkage includes both direct binding and linkage via a linker or spacer peptide. Preferably, "linked thereto" refers to a polypeptide comprising one, two, three, or four repeats of the peptide of SEQ ID NO: 1 linked to the C-terminus of 9 to 11 consecutive leucines.

[0025] That is, the present invention provides a polypeptide comprising 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked to the C-terminus thereof.

[0026] In the present invention, SEQ ID NO: 1 is a sequence known as NLS (nuclear localization sequence), which means a sequence composed of amino acids of PKKKRKV. This peptide does not simply function as an NLS, but rather is linked to 9, 10, or 11 consecutive leucines to achieve the desired effect of delivering a target substance into cells in a complex manner.

[0027] If necessary, the sequences may be linked directly or via a linker or spacer peptide between the peptides.

[0028] The term "linker" or "spacer" refers to a short amino acid sequence used to separate two functionally distinct peptides during polypeptide construction. The absence of a linker between two or more individual domains within a protein can result in reduced or inappropriate function of the protein domains due to steric hindrance, such as reduced catalytic activity or binding affinity for receptors / ligands. Linking protein domains in chimeric proteins using artificial linkers can increase the interdomain spacing. Preferably, the linker or spacer peptide is not particularly limited, as long as it exhibits the effect of improving the activity of the leucine-SEQ ID NO: 1 peptide bond or repeated SEQ ID NO: 1 peptide bond. While not possessing any specific biological activity other than connecting the domains or preserving some minimum distance or other spatial relationship between them, the constituent amino acids can be selected to affect certain properties of the molecule, such as folding, net charge, or hydrophobicity.

[0029] More specifically, the polypeptide may be any one selected from the group consisting of SEQ ID NOs: 2-13.

[0030] Such optional sequences are shown in Table 1 below.

[0031] [Table 1]

[0032] If necessary, the polypeptide sequence according to the present invention may consist of the above-mentioned SEQ ID NOs: 2 to 13. Here, peptides having at least 90% or more, most preferably 95%, 96%, 97%, 98%, or 99% or more sequence homology with any one selected from the group consisting of SEQ ID NOs: 2 to 13 are also included in the scope of the present invention.

[0033] Nine, ten, or eleven consecutive leucines are shown in SEQ ID NOs: 14, 15, and 16, respectively.

[0034] 9 consecutive leucines: LLLLLLLLL (SEQ ID NO: 14) 10 consecutive leucines: LLLLLLLLLL (SEQ ID NO: 15) 11 consecutive leucines: LLLLLLLLLLL (SEQ ID NO: 16)

[0035] In terms of sequence homology, the present invention may include polypeptides having a sequence that differs by one or more amino acid residues from any one selected from the group consisting of SEQ ID NOS: 2 to 13. Amino acid exchanges in proteins and polypeptides that do not change the overall activity of the molecule are known in the art. The most commonly performed exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. Furthermore, the present invention may include peptides whose structural stability against heat, pH, etc. is increased by mutations or modifications in the amino acid sequence.

[0036] The polypeptide according to the present invention is a polypeptide for use as a carrier capable of delivering a target substance into a cell.

[0037] The polypeptides according to the present invention fuse through direct interaction between the components, forming a common internal space in the fused particle. The target substance is carried in this common internal space and delivered into the cell. That is, the polypeptide can form a "membrane" to constitute an outer layer, which can carry the target substance by having an internal compartment. Specifically, the target substance may be carried by forming a membrane, which constitutes an outer layer, and having an internal compartment.

[0038] In particular, it has excellent physicochemical properties, variable fusion induction, high encapsulation efficiency, high safety, and low immunogenicity, making it highly applicable not only to cells but also to human applications.

[0039] Furthermore, since the polypeptide capable of delivering a target substance according to the present invention is a very small peptide, it is possible to minimize possible biological interference with the active substance.

[0040] Thus, in the present invention, a target substance can be delivered into a cell using a polypeptide.

[0041] In the present invention, the term "target substance" refers to any substance that can be carried by a polypeptide, delivered into a cell, and exhibits activity to regulate intracellular activity. Examples of the target substance include, but are not limited to, compounds, proteins, and nucleic acids.

[0042] More specifically, the compound may be a low molecular weight compound, a charged high molecular weight compound, or a fluorescent compound.

[0043] More specifically, the protein may be any one or more selected from the group consisting of an antibody, a ligand peptide capable of binding to a receptor, a protein drug, a cytotoxic polypeptide, a cytotoxic protein, and a fluorescent protein.

[0044] More specifically, the nucleic acid may be selected from the group consisting of, for example, DNA, recombinant DNA, plasmid DNA, antisense oligonucleotides, aptamers, RNA, siRNA, shRNA and miRNA.

[0045] The polypeptides according to the present invention can be produced using techniques known and available in the art, and can be synthesized using any suitable procedure known to those skilled in the art, i.e., known polypeptide synthesis methods (e.g., genetic engineering methods, chemical synthesis).

[0046] For example, the polypeptides of the present invention can be produced by recombinant techniques using genetic engineering methods. To produce a peptide using genetic engineering methods, for example, a nucleic acid (polynucleotide) encoding the polypeptide of the present invention or a functional equivalent thereof is first prepared by conventional methods. The nucleic acid can be prepared by PCR amplification using appropriate primers. Alternatively, a DNA sequence can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer. The prepared nucleic acid is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) operatively linked to the vector to regulate the expression of the nucleic acid to prepare a recombinant expression vector. The vector is then transformed into a host cell, and the cell is cultured in a medium and under conditions suitable for expression of the desired polypeptide. A substantially pure polypeptide expressed from the nucleic acid is then recovered from the culture. This recovery can be carried out using methods known in the art. Separation and purification can be performed by methods known in the art, including, but not limited to, extraction, recrystallization, various chromatographic techniques (gel filtration, ion exchange, precipitation, adsorption, reverse transcription), electrophoresis, and reverse current partitioning.

[0047] In the above, "substantially pure polypeptide" means that the polypeptide according to the present invention is substantially free from any other proteins derived from the host cell.

[0048] In the above, "vector" refers to a nucleic acid molecule capable of transporting a nucleic acid to a relevant site, and "expression vector" includes a plasmid, cosmid, or phage capable of synthesizing a fusion protein encoded by each recombinant gene carried by the vector.

[0049] For example, polypeptides according to the present invention can also be produced by chemical synthesis methods known in the art, including, but not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.

[0050] For example, the polypeptides of the present invention can be produced by direct peptide synthesis using the solid-phase peptide synthesis (SPPS) method. In this method, synthesis begins with attaching functional units called linkers to small porous beads, allowing peptide chains to be linked. Unlike in solution-phase synthesis, peptides are covalently bound to the beads, preventing their release through filtration until cleaved with a specific reactant such as trifluoroacetic acid (TFA). Synthesis is achieved through repeated cycles of protection, in which the N-terminal amine of the peptide attached to the solid phase is bonded to an N-protected amino acid unit, deprotection, and coupling, in which the exposed amine group is bonded to a new amino acid (deprotection-wash-coupling-wash). The SPPS method can be performed using microwave technology, which can shorten the time required for coupling and deprotection in each cycle by applying heat during peptide synthesis. The thermal energy can prevent the folding or aggregation of the expanding peptide chain and promote chemical bonding.

[0051] The present invention provides polynucleotides encoding the polypeptides.

[0052] Target substance delivery applications Another aspect of the present invention to achieve the above object provides a composition comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance.

[0053] Another aspect of the present invention to achieve the above-mentioned object provides a composition for intracellular transfection comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance.

[0054] Another aspect of the present invention to achieve the above object provides a composition for intranuclear delivery of a target substance, comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance.

[0055] The individual uses described above will now be described in detail.

[0056] The present invention provides a composition comprising a polypeptide consisting of 9, 10, or 11 consecutive leucine residues and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance. Such a composition can be used for intracellular transfection.

[0057] The composition of the present invention can exert various effects by transfecting a target substance into cells and regulating the expression of the target substance within the cells. The terms "transport," "infiltration," "transport," "transmission," "permeation," and "passage" are used interchangeably with "injection."

[0058] In the present invention, the term "transfection" refers to the process of injecting a nucleic acid molecule or protein into a cell, preferably a eukaryotic cell. The nucleic acid molecule may be a gene sequence encoding an entire protein or a functional portion thereof. The eukaryotic cell may be an animal cell, a mammalian cell, or a human cell, such as a stem cell (e.g., embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell, neural stem cell, mesenchymal stem cell, hematopoietic stem cell, peripheral blood stem cell), primary cell (e.g., myoblast, fibroblast), immune cell (e.g., NK cell, T cell, dendritic cell, antigen-presenting cell), cancer cell, epithelial cell, skin cell, gastrointestinal cell, mucosal cell, or lung cell.

[0059] The composition of the present invention increases the transfection efficiency of target cells, facilitating the delivery of target substances into target cells, and, if necessary, also enables delivery of therapeutic target substances into the nucleus.

[0060] The compositions of the present invention are intended for therapeutic, imaging, and diagnostic applications. Therefore, by using the polypeptides of the present invention, various target substances can be easily encapsulated in vitro or in vivo, preferably in the human body, and intracellular transfection of the target substances can be performed accordingly.

[0061] The polypeptides according to the present invention form spherical shapes through interactions between these molecules, and can carry target substances inside these spherical shapes. Such spherical shapes have diameters in the range of approximately 30 nm to 200 nm, more preferably 50 nm to 150 nm, and even more preferably 60 to 130 nm. The target substances can be contained inside these spherical shapes.

[0062] In particular, the hydrodynamic radius and zeta potential of the prepared polypeptide carrier were measured using a particle size analyzer, and the hydrodynamic radius was confirmed to be 30 nm to 50 nm, and the zeta potential was confirmed to be approximately +2 mV to +6 mV. This indicates that nano-sized complexes can be prepared using the polypeptide of the present invention and a target substance, and that the desired size is maintained regardless of whether the target substance is loaded or not. Furthermore, it was confirmed that the target substance can be loaded at a desired level or higher without affecting hybridization, demonstrating delivery efficacy.

[0063] For example, the polypeptide of the present invention can be used to introduce a target substance into cells by a general method well known in the art, which can be carried out within the level of commonly known cell culture conditions.

[0064] For example, the mixture can be mixed with a target substance in vitro and transfected into cells. The mixture can be performed at 32-40°C, preferably about 37°C, for 10, 20, 30, 40, 50, or 60 minutes, or for 2, 3, 4, 5, 6, or more hours. The buffer used for the mixture can be any well-known medium. Examples include, but are not limited to, Opti-MEM, DPBS, and / or RPMI-1640.

[0065] Furthermore, the polypeptide according to the present invention can be mixed with a target substance under ex vivo or in vivo conditions, thereby making the target substance accessible to target cells. In the present invention, transfection of the target substance can be carried out for 0.5, 1, 2, 3, 4, 5, or more hours.

[0066] In some embodiments, the composition (polypeptide and desired substance) may be pre-incubated to form a mixture of the polypeptide and desired substance prior to contacting the target cells.

[0067] The method can also include multiple treatments of the cells with the composition (e.g., 1, 2, 3, 4 or more times per day and / or on a predetermined schedule). In this case, a lower concentration of the composition may be recommended (e.g., for reduced toxicity). In some embodiments, the cells may be suspension cells or adherent cells. In some embodiments, those skilled in the art can adapt the teachings of this description using different combinations of carriers, domains, uses, and methods to suit the specific needs of delivering a substance of interest to specific cells with desired viability.

[0068] For example, the target substance may be a compound, a protein, a nucleic acid, or the like.

[0069] More specifically, the compound may be a low molecular weight compound, a charged high molecular weight compound, or a fluorescent compound.

[0070] More specifically, the protein may be any one or more selected from the group consisting of an antibody, a ligand peptide capable of binding to a receptor, a protein drug, a cytotoxic polypeptide, a cytotoxic protein, and a fluorescent protein.

[0071] More specifically, the nucleic acid may be selected from the group consisting of, for example, DNA, recombinant DNA, plasmid DNA, antisense oligonucleotides, aptamers, RNA, siRNA, shRNA and miRNA.

[0072] The target substance can be delivered into cells or the human body while changing the target substance depending on the purpose of treatment or prevention, experimental purpose for research and development, etc. Such a substance may be any substance for investigating various activities, such as endogenous ligands, neurotransmitters, hormones, autacoids, cytokines, antiviral agents, anticancer agents, antibiotics, oxygen-enhancing agents, oxygen-containing agents, antiepileptic agents, and anti-inflammatory drugs.

[0073] Transfection of a target substance using the composition according to the present invention can significantly increase the delivery efficiency into cells compared to conventional transfection. As a result, the composition according to the present invention can deliver a target substance into cells and exhibit therapeutic efficacy against diseases or disorders. According to one embodiment of the present invention, the polypeptide according to the present invention can deliver a large amount of a target substance into cells.

[0074] The present invention provides a method for delivering a target substance into a cell, which comprises the step of contacting the composition with the cell.

[0075] The cell may be an animal cell, a mammalian cell, or a human cell, such as, but not limited to, a stem cell (e.g., embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell, neural stem cell, mesenchymal stem cell, hematopoietic stem cell, peripheral blood stem cell), primary cell (e.g., myoblast, fibroblast), immune cell (e.g., NK cell, T cell, dendritic cell, antigen-presenting cell), cancer cell, epithelial cell, skin cell, gastrointestinal cell, mucosal cell, or lung cell.

[0076] therapeutic use Another aspect of the present invention to achieve the above object provides a composition for drug delivery comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance.

[0077] Another aspect of the present invention to achieve the above object provides a pharmaceutical adjuvant composition comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance.

[0078] Another aspect of the present invention to achieve the above object provides a composition for preventing or treating a disease, comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a drug.

[0079] Another aspect of the present invention to achieve the above object provides a composition for preventing or treating cancer, comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a target substance.

[0080] In the present invention, "for drug delivery" means use as a transmitter for delivering a drug into target cells.

[0081] In the present invention, the term "adjuvant use" refers to the use as an adjuvant in combination with a conventional drug to maximize the effect of the drug.

[0082] The drug delivery or drug adjuvant uses refer to uses that have relatively low medicinal effects when administered alone, but significantly improve the efficacy of a drug when administered together with the polypeptide of the present invention.

[0083] In the present invention, the composition can be used to deliver a target substance into biological tissue or blood. The composition can be delivered through cells or intercellular connections that constitute biological tissue, but the delivery method is not limited.

[0084] The biological tissue refers to one or more of epithelial tissue, muscle tissue, nerve tissue, and connective tissue, and each organ may consist of one or more tissues, and may include various biological organs such as mucous membrane, skin, brain, lung, liver, kidney, spleen, heart, gastrointestinal tract, large intestine, digestive tract, bladder, ureter, urethra, ovaries, testes, reproductive organs, muscle, blood, blood vessels, lymphatic vessels, lymph nodes, thymus, pancreas, adrenal gland, thyroid gland, parathyroid gland, larynx, tonsil, bronchi, and alveoli, but is not limited thereto.

[0085] In particular, the composition of the present invention not only delivers a target substance, e.g., a biologically active substance, into cells to allow it to act directly, but also targets one or more immune cells selected from the group consisting of macrophages, B lymphocytes, T lymphocytes, mast cells, monocytes, dendritic cells, eosinophils, natural killer cells, basophils, and neutrophils, and delivers the biologically active substance to act within the immune cells. Furthermore, unlike conventional techniques that could only deliver genes to immune cells via viral vectors, the composition of the present invention can deliver genes to immune cells via non-viral vectors, which may be a breakthrough in the development of drug delivery systems.

[0086] In the present invention, the target substance is, as mentioned above, a functional regulator that has biological activity that regulates all physiological phenomena in the body when carried by the polypeptide and delivered into cells, and means any substance that is to be delivered into cells.

[0087] The drug may be selected from the group consisting of, but not limited to, a compound drug, a biologic drug, a nucleic acid drug, a peptide drug, a protein drug, a hormone, a contrast agent, and an antibody. Preferably, the nucleic acid drug may be selected from the group consisting of DNA, recombinant DNA, plasmid DNA, antisense oligonucleotide, aptamer, RNA, siRNA, shRNA, and miRNA.

[0088] The drug according to the present invention may be a nucleic acid drug or an antibody.

[0089] Specifically, the substance may be one that is difficult to transfer into cells via a general pathway, or one that is easy to transfer into cells but has low specific transfer efficiency. More specifically, the substance may be an antibody that is difficult to transfer into most cells, or a genetic substance such as a plasmid, mRNA, or siRNA that is difficult to transfer into immune cells, stem cells, or nerve cells.

[0090] The present invention is applicable to methods for intracellular delivery of target substances (preferably drugs) into cells in vivo, which can be achieved by parenteral administration or direct injection into tissues, organs, or systems.

[0091] That is, the composition according to the present invention can be used in mammals, preferably humans, and can be administered, for example, intravenously, intraperitoneally, intramuscularly, subcutaneously, intradermal, nasal, mucosal, inhalation, or orally to deliver a target substance into cells.

[0092] In the present invention, the term "treatment" means suppression or alleviation of a disease or disorder. Therefore, in the present invention, the term "therapeutically effective amount" means an amount sufficient to achieve the pharmacological effect.

[0093] The composition can be provided by being formulated into an appropriate form, which can be formulated into oral dosage forms such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, and aerosols, or parenteral dosage forms such as transdermal agents, suppositories, and sterile injection solutions, by a conventional method.

[0094] In addition, the formulation may further contain auxiliaries such as pharmaceutically suitable and physiologically acceptable carriers, excipients, and diluents. Carriers, excipients, and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants can be used.

[0095] More specifically, the formulation may contain a carrier to be added to the composition (active ingredient) to form the composition into a dosage form, which may include a binder, lubricant, suspending agent, solubilizer, buffer, preservative, lubricant, isotonicity agent, excipient, stabilizer, dispersant, suspending agent, colorant, flavoring, etc.

[0096] The compositions can be administered alone, but may also be administered in admixture with a pharmaceutical carrier, generally selected having regard to the mode of administration and standard pharmaceutical practice.

[0097] For example, when the formulation is provided for parenteral administration, it may be in the form of a liquid dosage form such as a topical preparation (e.g., a solution, gel, cleansing composition, insert tablet, suppository, cream, ointment, dressing solution, spray, or other topical application), or a liquid dosage form such as a solution, suspension, or emulsion. It may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a suppository, a cream, ointment, jelly, foam, cleanser, or insert, preferably a topical preparation for skin (e.g., a solution, gel, cleansing composition, insert tablet). For example, the dosage form may be prepared by adding a solubilizing agent, an emulsifier, a buffer for pH adjustment, etc. to sterile water. Examples of the non-aqueous solvent or suspension solvent that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0098] Also, when the formulation is provided for oral use, it can be administered orally, bucally, or sublingually, for example, in purified form containing starch or lactose, or in capsule form alone or containing excipients, or in the form of an elixir or suspension containing flavoring or coloring chemicals.

[0099] The dosage of the formulation may vary depending on the patient's age, weight, sex, dosage form, health condition, and severity of the disease, and may be administered once or several times a day at predetermined time intervals according to the judgment of a doctor or pharmacist. For example, the daily dosage based on the active ingredient content may be 0.001 to 10,000 mg / kg, 0.01 to 10,000 mg / kg, 0.1 to 10,000 mg / kg, 0.5 to 10,000 mg / kg, 0.001 to 1,000 mg / kg, 0.01 to 1,000 mg / kg, 0.1 to 1,000 mg / kg, 0.5 to 1,000 mg / kg, 0.001 to 500 mg / kg, 0.01 to 500 mg / kg, 0.1 to 500 mg / kg, 0.5 to 500 mg / kg, 0.001 to 300 mg / kg, 0.01 to 300 mg / kg, 0.1 to 300 mg / kg, or 0.5 to 300 mg / kg. The above dosages are examples of average cases, and the dosage may be higher or lower depending on individual differences.

[0100] If the daily dose of the composition is less than the above-mentioned dosage volume, no significant effect will be obtained, and if it exceeds the above-mentioned dosage volume, not only will it be uneconomical but it may also deviate from the range of usual doses, and there may be a risk of undesirable side effects occurring, so it is preferable to keep it within the above-mentioned range.

[0101] The subject to which the composition is administered may be a mammal such as a human, or a cell, tissue, or body fluid isolated from a mammal, or a culture thereof.

[0102] The present invention also provides a composition for use in producing a genetically altered cell comprising a nucleic acid molecule encoding a chimeric antigen receptor, or a nucleic acid construct comprising the nucleic acid molecule, and a polypeptide consisting of 9, 10, or 11 consecutive leucines followed by 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1.

[0103] More specifically, the chimeric antigen receptors (CARs) described herein can be produced by any means known in the art, preferably using recombinant DNA technology. Nucleic acids encoding several regions of the chimeric receptor can be prepared and conveniently assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The obtained coding regions are preferably inserted into an expression vector and used to transform a suitable expression host cell line, an immune cell line, preferably a T lymphocyte cell line, and most preferably an autologous T lymphocyte cell line.

[0104] The nucleic acid constructs include expression vectors that contain a nucleic acid sequence encoding the chimeric antigen receptor described above.

[0105] The nucleic acid molecule can comprise any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified or modified, RNA or DNA. For example, the nucleic acid molecule can comprise single-stranded and / or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA that can be single-stranded, or more typically, double-stranded or a mixture of single- and double-stranded regions. Nucleic acid molecules can also contain triple-stranded regions containing RNA or DNA, or both RNA and DNA. Nucleic acid molecules can also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. Various modifications can be made to DNA and RNA, and the term "nucleic acid molecule" includes chemically, enzymatically, or metabolically modified forms.

[0106] It should be understood that the nucleic acid construct may further comprise one or more of the following: an origin of replication for one or more hosts; a selectable marker gene active in one or more hosts; and / or one or more transcriptional control sequences, wherein expression of the nucleic acid molecule is under the control of the transcriptional control sequences. As used herein, the term "selectable marker gene" includes any gene that confers a phenotype on cells in which it is expressed, thereby facilitating the identification and / or selection of cells transfected or transfected with the construct. A "selectable marker gene" includes any nucleotide sequence that, when expressed by cells transfected with the construct, confers a phenotype on cells that facilitates the identification and / or selection of such transfected cells. A wide variety of nucleotide sequences encoding suitable selectable markers are known in the art. Exemplary nucleotide sequences encoding selectable markers include the following: Among others, which allow optimal selection of cells using techniques such as Fluorescence-Activated Cell Sorting (FACS), are: adenosine deaminase (ADA) gene; cytosine deaminase (CDA) gene; dihydrofolate reductase (DHFR) gene; histidinol dehydrogenase (hisD) gene; puromycin-N-acetyltransferase (PAC) gene; thymidine kinase (TK) gene; xanthine-guanine phosphoribosyltransferase (XGPRT) gene or antibiotic resistance genes, such as ampicillin-resistance gene, puromycin-resistance gene, bleomycin-resistance gene, hydromycin-resistance gene, kanamycin-resistance gene and ampicillin-resistance gene; fluorescent reporter genes, such as green, red, yellow or blue fluorescent protein-encoding genes; and luminescence-based reporter genes, such as luciferase gene.

[0107] As set forth above, the nucleic acid construct may also include one or more transcription control sequences. As used herein, the term "transcription control sequence" may be understood to include any nucleic acid sequence that affects transcription of an operably linked nucleic acid. Such transcription control sequences may include, for example, a leader, a polyadenylation sequence, a promoter, an enhancer or upstream activating sequence, and a transcription terminator.

[0108] Typically, a transcription control sequence includes at least a promoter. As used herein, the term "promoter" refers to any nucleic acid that confers, activates, or enhances expression of a nucleic acid in a cell. A transcription control sequence is considered "operably linked" to a given nucleic acid molecule if the transcription control sequence is capable of promoting, repressing, or otherwise regulating transcription of the nucleic acid molecule.

[0109] The nucleic acid molecule is under the control of a transcriptional control sequence, for example, a constitutive promoter or an inducible promoter. A promoter can regulate the expression of an operably linked nucleic acid molecule constitutively or differentially in relation to the cell, tissue, or organ in which expression occurs. Thus, a promoter can include, for example, a constitutive promoter or an inducible promoter. A "constitutive promoter" is a promoter that is active under most environmental and physiological conditions. An "inducible promoter" is a promoter that is active under specific environmental or physiological conditions. The present invention contemplates the use of any promoter that is active in the target cell. Therefore, a wide range of promoters can be easily estimated by those skilled in the art. Mammalian constitutive promoters can include, but are not limited to, Simian virus 40 (SV40), cytomegalovirus (CMV), P-actin, ubiquitin C (UBC), elongation factor-1 alpha (E3A), phosphoglycerate kinase (PGK), and CMV early enhancer / chicken β-actin (CAGG). Inducible regulatory sequences can also include terminators. As used herein, the term "terminator" refers to a DNA sequence at the end of a transcription unit that signals the end of transcription. A terminator is a 3'-untranslated DNA sequence that typically contains a polyadenylation signal, which promotes the addition of polyadenylate sequences to the 3' end of the primary transcript. For use with promoter sequences, the terminator may be any terminator sequence that is operable in the cells, tissues, or organs in which it is intended to be used. Suitable terminators will be known to those skilled in the art.

[0110] Nucleic acid constructs according to the invention may further comprise additional sequences, for example sequences that allow for improved expression, cytoplasmic or membrane transport, and location signals.

[0111] The present invention essentially extends to all genetic constructs as described herein. Such constructs may further comprise nucleotide sequences intended for the maintenance and / or replication of the genetic construct in eukaryotic cells and / or for the integration of the genetic construct or parts thereof into the genome of a eukaryotic cell. The nucleic acid construct may be in any suitable form, such as a plasmid, phage, transposon, cosmid, chromosome, vector, etc., which, when associated with appropriate regulatory elements, is capable of replication and transfer of the genetic sequences contained within the construct between cells.

[0112] In at least some embodiments, the present invention provides nucleic acid molecules, or nucleic acid constructs, encoding the above-described CARs for use in producing genetically altered cells.

[0113] In at least some embodiments, the present invention also provides for the use of nucleic acid molecules in the preparation of vectors for the transformation, transfection, or transfection of cells. Preferably, the cells are T cells that express one or more of CD3, CD4, or CD8. Cells suitable for genetic modification may be xenogeneic or autologous.

[0114] The nucleic acid molecule encoding the chimeric antigen receptor described above, or a nucleic acid construct containing the nucleic acid molecule, can be injected into cells via the polypeptide of the present invention. Such intracellular introduction can achieve intracellular transfection with higher efficiency than known intracellular transfection methods, and can provide cells transformed with a CAR that exhibits superior therapeutic efficacy.

[0115] Here, the present invention provides a composition for preventing or treating cancer, which comprises a polypeptide consisting of 9, 10, or 11 consecutive leucine residues and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a nucleic acid molecule encoding a chimeric antigen receptor, or a nucleic acid construct containing the nucleic acid molecule.

[0116] More specifically, the present invention provides a composition for preventing or treating cancer, which comprises a polypeptide consisting of 9, 10, or 11 consecutive leucine residues and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a nucleic acid molecule encoding a chimeric antigen receptor, or a cell genetically transformed with a nucleic acid construct containing the nucleic acid molecule.

[0117] That is, the present invention provides the use of genetically modified cells as described above for preventing or treating cancer. Accordingly, the present invention provides a method for preventing or treating a patient with cancer, such method comprising the step of exposing the patient to cells expressing a chimeric antigen receptor.

[0118] Here, the cells may preferably be T cells or NK cells, and more preferably, the cells are T cells that express CD3, CD4, or CD8.

[0119] The cancer may be any one or more selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, epithelial cancer, esophageal cancer, lung cancer, mouth cancer, ovarian cancer, kidney cancer, liver cancer, leukemia, lymphoma, myeloma, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, and tongue cancer.

[0120] The therapeutic composition may be administered about once to about five times per week. In some embodiments, the composition is administered once. In some embodiments, the composition is administered twice. In some embodiments, the composition is administered three times. In some embodiments, the composition is administered four times. In some embodiments, the composition is administered in a dose of at least 5×10 8 Contains cells.

[0121] According to an embodiment of the present invention, CAR-T cells were produced by carrying a CAR DNA plasmid in a polypeptide according to the present invention, and their therapeutic efficacy against cancer was confirmed. Generally, CAR-T cells have poor transformation efficiency. However, when the polypeptide according to the present invention is used as a delivery system, it exhibits high delivery efficiency for plasmid DNA, enabling mass production of CAR-T cells with excellent therapeutic efficacy.

[0122] The present invention provides a method for treating or preventing a disease or disorder, comprising the step of administering a therapeutically effective amount of the composition to a subject in need thereof.

[0123] The present invention provides a method for delivering a target substance into a cell, which comprises the step of treating the cell with the composition.

[0124] The present invention provides a method for delivering a target substance into a cell, which comprises the step of administering to a subject a polypeptide comprising the composition.

[0125] The present invention provides a method for selectively delivering a target substance into a cell, which comprises the step of treating the cell with the composition.

[0126] The present invention provides a method for selectively delivering a target substance into cells, which comprises the step of treating a subject with the composition.

[0127] The present invention provides compositions comprising the desired substance for use in treating or preventing the above diseases or disorders.

[0128] The present invention provides a composition comprising a polypeptide for delivering a target substance into a cell.

[0129] The present invention provides use of a polypeptide in the production of a preparation for delivering a target substance into a cell.

[0130] The present invention also provides uses and methods of utilizing the compositions described above. [Effects of the Invention]

[0131] The novel peptide composition for intracellular transfection according to the present invention has the advantages of significantly improving the transfection efficacy of target substances and significantly reducing cytotoxicity, making it useful for transporting various substances into cells. [Brief explanation of the drawings]

[0132] [Figure 1] FIG. 1 shows the efficiency of plasmid DNA transfer into Jurkat T cells for peptides containing various hydrophobic amino acids (Peptides 1 to 10). [Figure 2] FIG. 1 shows the efficiency of plasmid DNA transfer into Jurkat T cells for peptides with different leucine lengths and 10 or 11 leucines (Peptides 1 and 11). [Figure 3] This figure shows the efficiency of plasmid DNA transfer into Jurkat T cells for peptides (Peptides 1, 12, and 13) prepared with different NLS copy numbers (2, 3, or 4). [Figure 4] FIG. 1 shows the efficiency of plasmid DNA transfer into Jurkat T cells for peptides in which the NLS was positioned at the C-terminus or N-terminus (Peptides 1 and 14). [Figure 5] FIG. 1 shows changes in GFP expression depending on L10-2xNLS concentration, confirmed by immunoblotting. [Figure 6] FIG. 1 shows changes in GFP expression depending on the concentration of pEGFP-N3, confirmed by immunoblotting. [Figure 7] FIG. 1 shows changes in GFP expression depending on the temperature at which L10-2xNLS and pEGFP-N3 are mixed, as confirmed by immunoblotting. [Figure 8] FIG. 1 shows changes in GFP expression depending on the time for mixing L10-2xNLS and pEGFP-N3, confirmed by immunoblotting. [Figure 9] FIG. 1 shows the change in GFP expression depending on the type of buffer used to mix L10-2xNLS and pEGFP-N3, as confirmed by immunoblotting. [Figure 10] FIG. 1 shows changes in GFP expression depending on the incubation time of transfection, as confirmed by immunoblotting. [Figure 11] FIG. 1 shows the size of the complex between L10-2xNLS and plasmid DNA. [Figure 12] FIG. 10 is a graph confirming the zeta potential of the complex of L10-2xNLS and plasmid DNA. [Figure 13] FIG. 1 shows the shape of the complex of L10-2xNLS and plasmid DNA confirmed by transmission electron microscopy (TEM). [Figure 14] This figure confirms the change in shape of Jurkat T cells after transfection with plasmid DNA using L10-2xNLS. [Figure 15] FIG. 1 shows changes in viability of Jurkat T cells after transfection with plasmid DNA using L10-2xNLS. [Figure 16] FIG. 10 shows the efficiency of plasmid DNA transfer into Jurkat T cells using L10-2xNLS. [Figure 17]FIG. 1 shows the location of plasmid DNA delivered using L10-2xNLS in Jurkat T cells. [Figure 18] FIG. 1 shows the quantification of the location of plasmid DNA delivered using L10-2xNLS in Jurkat T cells. [Figure 19] FIG. 1 shows a graph confirming the gene transfer effect of L10-2xNLS on human primary T cells. [Figure 20] This figure confirms the activity efficacy of CAR-T manufactured using L10-2xNLS. [Figure 21] FIG. 10 shows the antibody transduction effect on Jurkat T cells confirmed using L10-2xNLS. [Figure 22] FIG. 10 shows a graph confirming the presence or absence of antibodies within Jurkat T cells using L10-2xNLS. DETAILED DESCRIPTION OF THE INVENTION

[0133] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention and are not intended to limit the scope of the present invention.

[0134] Example 1. Amino acid sequences of peptides optimized for plasmid DNA delivery 1-1. Plasmid DNA transfer effect depending on the type of hydrophobic amino acid The peptides were prepared by conjugating a peptide consisting of two copies of the nucleus localization sequence (NLS, PKKKRKV) (2Xnls) to a sequence containing leucine (L), phenylalanine (F), methionine (M), valine (V), glycine (g), proline (p), alanine (A), tyrosine (Y), isoleucine (I), and / or tryptophan (W).

[0135] The specific sequences are shown in Table 2 below (Peptides 1-10). Each peptide was mixed at a concentration of 4 μM with 2 μg of pEGFP-N3 plasmid for 30 minutes in a CO2 incubator at 37°C. Jurkat T cells were transfected with the plasmid for 30 minutes, and GFP expression was analyzed 24 hours later using immunoblotting (Figure 1).

[0136] As a result, L, which is composed of leucine, 10 The -2xNLS peptide was found to induce the highest GFP expression, and this transfection showed significantly higher levels than lipofectamine, which is commonly used for transfection.

[0137] 1-2. Plasmid DNA transfer efficiency depending on the length of leucine amino acids Peptides containing 10 or 11 leucine amino acids and 2xNLS were prepared (Peptides 1 and 11), and the effect of the number of leucine amino acids on permeability was further confirmed. The peptides were subjected to the same experiment as in Example 1-1, and GFP expression was analyzed by immunoblotting (Figure 2).

[0138] As a result, it was confirmed that the highest efficiency was observed when the leucine length was 10, and that even when the leucine length was 11, the efficiency remained above the predetermined level.

[0139] 1-3. Plasmid DNA delivery efficiency depending on NLS copy number and N-terminal / C-terminal position Peptides were prepared by varying the number of copies of the NLS located after the 10 leucine amino acid sequence (2, 3, or 4), and the specific sequences are shown in Table 2 below (Peptides 1, 12, and 13). For each peptide, immunoblotting was used to confirm the change in GFP expression depending on the copy number of the NLS (Figure 3).

[0140] As a result, the highest GFP expression was observed when pEGFP-N3 was transfected into Jurkat T cells using L10-2xNLS. Furthermore, it was confirmed that the expression level was maintained above the specified level even when the NLS sequence was increased to three or four.

[0141] As a result, the copy number of NLS was set to 2, and a peptide (L 10 -2xNLS), and a peptide located at the N-terminus in front of leucine (2xNLS-L 10 The peptides (Peptides 1 and 14) were mixed with pEGFP-N3 and transfected into Jurkat T cells, and the changes in GFP expression were compared. The transfection efficiency was confirmed, and it was found that the peptide (L) in which the NLS amino acid sequence was located at the C-terminus after leucine was the most efficient. 10 -2xNLS) showed high efficiency (Figure 4).

[0142] That is, the peptide optimized for plasmid DNA delivery to Jurkat T cells contains approximately 10 leucine amino acid sequences and was confirmed to exhibit transfection efficacy above a certain level at 2-4 copies of NLS. 10 It was confirmed that -2xNLS contains the amino acid sequence of a peptide optimized for plasmid DNA delivery to Jurkat T cells.

[0143] The specific sequences of Peptides 1 to 14 used in Example 1 are summarized in Table 2 below. Each peptide was modified by N-terminal acetylation or C-terminal amidation to increase the stability and bioactivity of the peptide.

[0144] [Table 2]

[0145] Example 2. Optimized conditions for intracellular transfection of plasmid DNA 2-1. Plasmid DNA transfer efficiency depending on the concentration of L10-2xNLS and pEGFP-N3 L 10 2xNLS was mixed with 2 μg of pEGFP-N3 at different concentrations (2, 3, 4, 5, or 6 μM) and transfected into Jurkat T cells, and GFP expression was confirmed by immunoblotting (Figure 5).

[0146] Result, L 10 It was found that the highest expression efficiency of GFP was observed when the concentration of -2xNLS was 4 μM.

[0147] Next, pEGFP-N3 was added at different concentrations (0, 0.25, 0.5, 1, 2, 4, 5, or 6 μg) to 4 μM L 10 The resulting mixture was mixed with 2xNLS and transfected into Jurkat T cells. GFP expression was then confirmed by immunoblotting (Figure 6).

[0148] As a result, the highest GFP expression efficiency was observed when pEGFP-N3 was used at 2 μg.

[0149] Considering the above-mentioned target concentration range for transfection injection, the following experiment was carried out.

[0150] 2-2. Plasmid DNA transfer efficiency under mixed conditions of L10-2xNLS and pEGFP-N3 To find the most effective mixing conditions for plasmid DNA transfer, 4 μm L 10 The temperature, time, and buffer used for mixing 2xNLS and 2 μg of pEGFP-N3 were varied, and the gene transfer effect under each condition was compared.

[0151] First, 4 μm L was incubated at 4, 16, 25, 37, or 42 °C. 102xNLS and 2 μg of pEGFP-N3 were mixed for 30 minutes and transfected into Jurkat T cells for 30 minutes. After 24 hours, immunoblotting was used to analyze changes in GFP expression. The highest GFP expression was observed when the temperature during mixing was 37°C (Figure 7). This indicates that the system of the present invention works well under normal cell culture conditions and temperature conditions suitable for human applications.

[0152] This allows the temperature to be set at 37°C and the L of 4 µm 10 2xNLS and 2 μg of pEGFP-N3 were mixed for different times (10, 20, 30, 40, 50, or 60 minutes). The GFP expression was analyzed using the same method as above. The highest GFP expression level was observed after 30 minutes of mixing (Figure 8). This demonstrates the advantage of short-term transfection.

[0153] Finally, the buffer used for mixing was Opti-MEM, DPBS, or Serum-free rPi-1640 (SF), respectively, and the mixture was diluted to 4 μm L. 10 2xNLS was mixed with 2 μg of pEGFP-N3. The GFP expression was analyzed using the same method as above, and the results confirmed that the transfection level was maintained under various conditions. This confirmed that the system of the present invention can be applied and used under various culture conditions (Figure 9).

[0154] Considering the above-optimized temperature, time, and conditions, the following experiments were carried out.

[0155] 2-3. Plasmid DNA transfer efficiency depending on transfection time According to the optimized conditions, L of 4 μm 10 After mixing 2xNLS and 2 μg of pEGFP-N3, Jurkat T cells were transfected for 0.5, 1, 2, 3, 4, or 5 hours, and the changes in GFP expression over time were compared (FIG. 10).

[0156] As a result, it was confirmed that transfection for 0.5 or 1 hour was sufficient to transfer the plasmid DNA.

[0157] Example 3. Physicochemical characterization of L10-2xNLS and plasmid DNA complexes Using a particle size analyzer Zetasizer (Malvern Panalytical) to measure particle size, L 10 -2xNLS only mixture (L 10 -2xNLS only) and L 10 -2xNLS and pEGFP-N3 mixture (L 10 The size and zeta potential of the 2×NLS+pEGFP (2 μg) were analyzed (FIGS. 11 and 12).

[0158] Result, L 10 The size of the complex of 2xNLS and plasmid DNA is approximately 100 nm, and the zeta potential is L 10 It was confirmed that the ions were positively charged by +2 to +6 mV depending on the concentration of -2xNLS.

[0159] In addition, a transmission electron microscope (TEM) was used to 10 The morphology of the complex between 2xNLS and plasmid DNA was analyzed (FIG. 13). As a result, the complex was confirmed to have an oval shape with a size of 60 to 100 nm, as shown in FIG.

[0160] This allows L 10 It was confirmed that -2xNLS and plasmid DNA can form nano-sized complexes, and that the polypeptide according to the present invention can form spheres, carry nucleic acids within the spheres, and act as a transmitter.

[0161] Example 4. Effect of transfection with L10-2xNLS on cells 4.1. Damage and death of Jurkat T cells 4 μm L 10Jurkat T cells were transfected with a mixture of 2xNLS and 2 μg of pEGFP-N3. After 24 hours, changes in cell shape were observed (Figure 14). Cell viability after transfection was also analyzed using an MTT assay (Figure 15).

[0162] Result, L 10 -2xNLS was used to confirm that transfection into Jurkat T cells did not damage or kill the cells.

[0163] 4.2. Transduction efficiency and location analysis of Jurkat T cells 2 μg of fluorescently labeled plasmid DNA (Takara) was added to the L 10 The plasmid DNA was mixed with 4 μM of 2xNLS and transfected into Jurkat T cells for 0.5, 1, 2, 3, 4, or 5 hours. The transduction efficiency of the plasmid DNA into Jurkat T cells was then analyzed using Novocyte FACS (Agilent) (Figure 16).

[0164] As a result, lipofectamine showed only about 15% efficiency even after 5 hours. 10 -2xNLS delivered the plasmid DNA to most of the cells with approximately 90% efficiency even after 0.5 hours.

[0165] Next, fluorescently labeled plasmid DNA and L 10 The plasmid DNA was mixed with 2xNLS and transfected into Jurkat T cells, which were then treated with Hoechst 33342 (ThermoFisher), a nuclear staining reagent. The location of the plasmid DNA within Jurkat T cells was then confirmed using a fluorescence microscope (Leica) (Figure 17), quantified, and analyzed graphically (Figure 18). 10After transfection with -2xNLS, the plasmid DNA was delivered to most of the cells within 1 hour, and after 4 hours, the plasmid DNA was present in the nuclei of the cells with approximately 40% efficiency.

[0166] This confirms that the transfection system of the present invention can significantly improve intracellular delivery efficiency compared to conventional transfection methods.

[0167] Example 5. Intracellular delivery of plasmid DNA using L10-2xNLS and its efficacy 5-1. CAR-T's efficacy in killing blood cancer cells L 10 4 μm of 2xNLS and 2 μg of FLAG-tagged CAR plasmid DNA were mixed and transfected into human primary T cells. After 24 hours, CAR expression in the human primary CD8+ T cells was confirmed by immunoblotting using a FLAG antibody (Figure 19).

[0168] To confirm whether the CAR-T cells produced by the above method can kill blood cancer cells, CAR-T cells were cultured with luciferase-expressing blood cancer cells (Nalm6 cells) at a ratio of 10:1. After 6 hours, luciferase activity was measured, confirming that the CAR-T cells were effective in killing blood cancer cells (Figure 20).

[0169] From the above results, it was confirmed that the transfection system according to the present invention can deliver therapeutic substances in a target-specific manner and enhance cell killing efficacy compared to conventional transfection methods.

[0170] Example 6. Confirmation of antibody transduction efficacy using L10-2xNLS In order to confirm the possibility of delivery of not only nucleic acids but also other antibody drugs as delivery substances, 10The antibody was treated with 2xNLS to check for changes in transfection levels.

[0171] 6-1.Efficiency of antibody delivery to Jurkat T cells 1 μg of fluorescently labeled IgG (Thermofisher) was added to the L 10 Jurkat T cells were treated with each mixture for 2 hours, and the antibody delivery efficiency into the cells was confirmed using Novocyte FACS (Agilent) (Figure 21). The presence or absence of the antibody in the cells was observed using a fluorescence microscope (Leica) (Figure 22).

[0172] Result, L 10 When the concentration of -2xNLS was 4 μM or higher, the antibody was delivered into Jurkat T cells with approximately 90% efficiency.

[0173] In summary, L 10 It was confirmed that plasmid DNA or antibodies can be effectively delivered into cells using -2xNLS.

[0174] This confirmed that it is possible to deliver targeted polynucleotides, antibodies, etc. to various cells in a target-specific manner, and that this method is particularly applicable to the development of immune cell therapy agents such as CAR-T cells.

Claims

1. A polypeptide comprising 9, 10 or 11 consecutive leucines and 1, 2, 3 or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto.

2. The polypeptide of claim 1, wherein the peptide of sequence number 1 is linked to the C-terminus of leucine.

3. 2. The polypeptide of claim 1, comprising 10 or 11 consecutive leucines and 2, 3 or 4 repeated peptides of sequence number 1 linked thereto.

4. The polypeptide of claim 1, wherein the polypeptide is any one selected from the group consisting of SEQ ID NOs: 2 to 13.

5. The polypeptide of claim 4, wherein the polypeptide is SEQ ID NO: 7, 8, 11 or 12.

6. A composition for intracellular transfection comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of sequence number 1 linked thereto, and a target substance.

7. The composition for intracellular transfection according to claim 6 , wherein the target substance is a compound, a protein, or a nucleic acid.

8. The composition for intracellular transfection according to claim 7, wherein the protein is at least one selected from the group consisting of an antibody, a ligand peptide capable of binding to a receptor, a protein drug, a cytotoxic polypeptide, a cytotoxic protein, and a fluorescent protein.

9. The composition for intracellular transfection according to claim 7, wherein the nucleic acid is selected from the group consisting of DNA, recombinant DNA, plasmid DNA, antisense oligonucleotides, aptamers, RNA, siRNA, shRNA and miRNA.

10. The composition for intracellular transfection according to claim 6, wherein the polypeptide is any one selected from the group consisting of SEQ ID NOs: 2 to 13.

11. The composition for intracellular transfection according to claim 6, wherein the polypeptide forms a membrane to constitute an outer layer and has an internal compartment, thereby carrying a target substance.

12. The composition for intracellular transfection according to claim 6, wherein the cells are any one selected from the group consisting of stem cells, primary cells, immune cells, cancer cells, epithelial cells, skin cells, gastrointestinal cells, mucosal cells, and lung cells.

13. A composition for drug delivery comprising a polypeptide consisting of 9, 10 or 11 consecutive leucines and 1, 2, 3 or 4 repeats of the peptide of sequence number 1 linked thereto, and a target substance.

14. The drug delivery composition according to claim 13, wherein the drug is any one selected from the group consisting of a compound drug, a biodrug, a nucleic acid drug, a peptide drug, a protein drug, a hormone, a contrast agent, and an antibody.

15. The composition for drug delivery according to claim 13, wherein the polypeptide is any one selected from the group consisting of SEQ ID NOs: 2 to 13.

16. The drug delivery composition according to claim 13, wherein the polypeptide forms a membrane to constitute an outer layer and has an internal compartment, thereby carrying a target substance.

17. A composition for use in producing a genetically modified cell, comprising a polypeptide consisting of 9, 10, or 11 consecutive leucines and 1, 2, 3, or 4 repeats of the peptide of SEQ ID NO: 1 linked thereto, and a nucleic acid molecule encoding a chimeric antigen receptor, or a nucleic acid construct comprising the nucleic acid molecule.

18. 18. The composition of claim 17, wherein the cell is an immune cell.

19. A cell genetically modified according to claim 17 or claim 18.

20. A composition for preventing or treating cancer, comprising the genetically altered cells of claim 19.

21. The cancers include bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, and epithelial cancer. cancer), esophageal cancer, lung cancer, mouth cancer, ovarian cancer, kidney cancer, liver cancer cancer, leukemia, lymphoma, myeloma, pancreatic cancer, prostate cancer, rectal cancer, skin cancer 21. The composition for preventing or treating cancer according to claim 20, wherein the cancer is any one or more selected from the group consisting of breast cancer, stomach cancer, testicular cancer, thyroid cancer, and tongue cancer.

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