Nanoparticles containing peptide-based conjugates for delivery of mRNA into B and T cells and uses thereof

Abstract

The present invention relates to a peptide-based conjugate for mRNA delivery that overcomes the limitations of existing lipid nanoparticles and enables safe and immediate response to new variant infectious diseases. mRNA binds to the RNA-binding peptide of the conjugate, and self-assembly proceeds via amphiphilic polypeptides or polymers to form peptide and mRNA complex nanoparticles. It has been confirmed that the nanoparticles and pharmaceutical compositions containing the same efficiently increase intracellular delivery of mRNA, thereby demonstrating mRNA-based vaccine effects.

Description

[Technical Field]

[0001] The present invention relates to a peptide-based mRNA carrier that overcomes the limitations of existing lipid nanoparticles (LNPs) and can safely and immediately respond to new strains of infectious diseases. [Background technology]

[0002] Oligonucleotides, particularly in the form of siRNA and mRNA, are being developed as therapeutic agents for controlling genes that cause cancer and infectious diseases. Recently, the FDA approved an mRNA vaccine, which induces an immune response by injecting the gene encoding the spike protein of the COVID-19 virus in the form of mRNA. The application of mRNA has been attracting attention. Since mRNA is artificially synthesized from mRNA within the body, it is non-toxic. However, it is rapidly degraded by nucleases in the body and carries a negative charge, making it impossible to target specific lesions. Furthermore, while its effects are achieved by acting within the cytoplasm, its negative charge and large size prevent it from penetrating the cell membrane by itself. Therefore, to overcome these limitations, a vehicle capable of stably delivering mRNA into cells is needed.

[0003] The most commonly used delivery vehicles are lipid nanoparticles, and oligonucleotide delivery vehicles using a variety of cationic lipids are known (US 2006 / 0083780, US 2006 / 0240554, US 2008 / 0020058, US 2009 / 0263407, and US 2009 / 0285881, and WO 2009 / 086558, WO 2009 / 127060, WO 2009 / 132131, WO 2010 / 042877, WO 2010 / 054384, WO 2010 / 054401, WO 2010 / 054405, WO 2010 / 054406, and WO 2010 / 105209). Traditional cationic lipids such as CLinDMA and DLinDMA have been utilized for oligonucleotide delivery to the liver, but are known to experience non-optimal delivery efficiency along with hepatotoxicity at high doses.

[0004] Therefore, in order to prepare nanoparticles composed of a carrier peptide that can safely and effectively deliver an infectious disease mRNA vaccine into cells, the inventors prepared a peptide composed of a sequence for target cell surface recognition, cell penetrating functionality, and a sequence that can bind to infectious disease mRNA. They confirmed that when nanoparticles composed of a carrier peptide are used, mRNA is effectively delivered into cells and the loaded mRNA exerts a vaccine effect, thereby completing the present invention.

[0005] The above information provided in this Background Art section is merely intended to enhance understanding of the background of the present invention, and therefore may not include information that constitutes prior art already known to those skilled in the art to which the present invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a peptide-based conjugate that can safely and effectively deliver mRNA into cells.

[0007] Another object of the present invention is to provide nanoparticles for intracellular delivery of mRNA, which are formed by binding the peptide-based conjugate to mRNA, and a composition for intracellular delivery of mRNA, which contains the nanoparticles, for B cell or T cell delivery.

[0008] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating a viral infection, comprising the nanoparticles.

[0009] It is still another object of the present invention to provide a method for preventing or treating a viral infection, which comprises administering the nanoparticles.

[0010] It is still another object of the present invention to provide a use of the above nanoparticles for the prevention or treatment of viral infections.

[0011] Yet another object of the present invention is to provide a use of the above nanoparticles for the manufacture of a medicament for the prevention or treatment of viral infections. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides a peptide-based conjugate for mRNA delivery, which has the following structural formula 1 as a base unit: [Structural formula 1] ABDE Here, A is an RNA-binding peptide, B is an amphipathic polypeptide or amphipathic polymer, D is a peptide with cell-penetrating function, and E is a peptide that recognizes the surface of B cells or T cells.

[0013] The present invention also provides nanoparticles to which the peptide-based conjugate and mRNA are bound.

[0014] The present invention also provides a composition for delivering mRNA into B cells or T cells, comprising nanoparticles to which the peptide-based conjugate and mRNA are bound.

[0015] The present invention also provides a pharmaceutical composition for preventing or treating a viral infection, comprising nanoparticles to which the peptide-based conjugate and mRNA are bound.

[0016] The present invention also provides a method for preventing or treating a viral infection, comprising administering the nanoparticles.

[0017] The present invention also provides a use of the nanoparticles for the prevention or treatment of viral infections.

[0018] The present invention also provides use of the nanoparticles described above for the manufacture of a medicament for the prevention or treatment of a viral infection. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a peptide-based conjugate. [Figure 2] This is a schematic diagram showing the process in which mRNA first binds to the RNA-binding peptide of a peptide-based conjugate, and then nanoparticles are formed by self-assembly. [Figure 3] Figure 3A shows the results of electrophoresis on agarose gel after dissociation of nanoparticles composed of mRNA and peptide-based conjugates at each reaction ratio with 0.05% SDS. Figure 3B shows the results of observation of the formed nanoparticles using TEM. Figure 3C shows the size and distribution of nanoparticles using an electrophoretic light scattering spectrophotometer. [Figure 4] This is the result of observing the degree of penetration into cells when cells were treated with nanoparticles composed of mRNA-lipofectamine conjugate and mRNA-peptide conjugate. [Figure 5]The figure shows the results of Western blots observing the amount of intracellular protein expression caused by nanoparticles composed of mRNA and peptide conjugates, and the results of Western blots confirming that active KRAS is reduced by the protein expressed by mRNA after cells are treated with nanoparticles composed of mRNA and peptide conjugates. [Figure 6] B cell / T cell The binding of a target peptide to B cells / T cells was observed using a confocal microscope. [Figure 7] This is the result of observing the binding of a target peptide to B cells / T cells using flow cytometry. [Figure 8] TEM observation results of nanoparticles made with COVID-19 vaccine mRNA and peptide carriers. [Figure 9] These are the results of an antibody analysis against SARS-CoV-2 Spike Glycoprotein S1 formed by nanoparticles made with COVID-19 vaccine mRNA and peptide carriers. [Figure 10] These are the results of an analysis of the immune response formed by nanoparticles made with COVID-19 vaccine mRNA and peptide carriers. [Figure 11] These are the results of an analysis of human ACE2 antibodies formed by nanoparticles made with COVID-19 vaccine mRNA and peptide carriers. DETAILED DESCRIPTION OF THE INVENTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0021] In the present invention, we have prepared nanoparticles composed of peptide-based conjugates that can safely and efficiently deliver mRNA into cells. First, we prepared a peptide-based conjugate using a peptide composed of a sequence for target cell surface recognition, cell penetration functionality, endosomal escape promotion, and a sequence capable of binding to oligonucleotides, and an amphipathic polypeptide capable of protecting mRNA. We then prepared nanoparticles by conjugating mRNA to the above conjugate. It was confirmed that nanoparticles composed of the above conjugates effectively delivered mRNA into cells, and that the expression of the loaded mRNA demonstrated a vaccine effect against viral infection.

[0022] Therefore, in one aspect, the present invention relates to a peptide-based conjugate for mRNA delivery, which has the following structural formula 1 as a base unit: [Structural formula 1] ABDE where A is an RNA-binding peptide, B is an amphiphilic polypeptide or amphiphilic polymer; D is a peptide with cell-penetrating function, E is characterized as being a peptide that recognizes the surface of B cells or T cells.

[0023] In the present invention, A may be characterized by including a peptide sequence to which RNA binds, and may be characterized by being a peptide represented by any one of the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 15, but is not limited thereto. SEQ ID NO: 1: LKKLLKLLKKLLKLAG SEQ ID NO: 2: IKKLIKIIKKLIKLAG SEQ ID NO: 3: LRRLLRLLRRLLRLAG SEQ ID NO: 4: LKKLLKLLOrnKLLDprLAG SEQ ID NO: 5: LRRLLRLLOrnRLLDprLAG SEQ ID NO: 6: LRKIIRLIOrnKLLDprLAG SEQ ID NO: 7: LKKLLKLLOrnKLLKLAG SEQ ID NO: 8: WKKLLKLLKKLLKLAG SEQ ID NO: 9: WRRLLRLLRRLLRLAG SEQ ID NO: 10: WRKLLRLLKKLLKLAG SEQ ID NO: 11: LKKLLDbuLLKKLLKWAG SEQ ID NO: 12: LRRLLDbuLLRRLLRWAG SEQ ID NO: 13: LKRLIDbuIIKKLIKWAG SEQ ID NO: 14: LKKLLKWLOrnKLLDprLAG SEQ ID NO: 15: LRRLLRWLOrnRLLDbuLAG Orn = Ornithine, Dbu = 2,4-diaminobutyric acid, Dpr = 2,3-diaminopropionic acid.

[0024] In the present invention, A can be any peptide capable of binding to RNA, and for example, the nucleic acid-binding peptides disclosed in US 2020 / 0207834 A1 can be used, but are not limited thereto.

[0025] In the present invention, the above-mentioned B can be characterized as being an amphiphilic polypeptide or an amphiphilic polymer.

[0026] In the present invention, the term "amphipathic" is meant to include hydrophilic and hydrophobic domains.

[0027] The amphipathic polypeptide refers to a polypeptide containing polar and nonpolar amino acids, with the polar and nonpolar amino acids partially clustered together to form a polar portion and a nonpolar portion. The polar amino acids may include cysteine, glutamine, threonine, tyrosine, serine, or asparagine, and the nonpolar amino acids may include, but are not limited to, phenylalanine, tryptophan, methionine, proline, valine, isoleucine, leucine, glycine, or alanine.

[0028] In the present invention, the amphipathic polypeptide may be in a form in which polar amino acids and nonpolar amino acids are closely packed together in the amino acid sequence, or in a form in which polar amino acids and nonpolar amino acids are not closely packed together in the amino acid sequence but are closely packed together in the three-dimensional structure when a peptide containing the amino acids forms a three-dimensional structure, but is not limited thereto. For example, the amphipathic polypeptide may be in a form in which a plurality of identical or non-identical polar amino acids are bound to a plurality of identical or non-identical nonpolar amino acids.

[0029] In one embodiment of the present invention, the amphipathic polypeptide may be a peptide represented by the amino acid sequence of SEQ ID NO: 16, but is not limited thereto. SEQ ID NO: 16: PLVFNQER

[0030] The amphiphilic polymer is a polymer formed by binding a hydrophobic polymer and a hydrophilic polymer, and is well known to those skilled in the art. The amphiphilic polymer is a polymer bound in a hydrophilic-hydrophobic form, and may include, but is not limited to, poloxamer, polyethylene oxide (PEO)-polylactic acid (PLA) copolymer, or a form in which the hydrophilic amino acid RALA is bound to the hydrophobic polymer polyetherimide (PEI). The amphiphilic polymer may be selected according to the application and purpose of the self-associating amphiphilic polymer. In one embodiment of the present invention, the amphiphilic polymer may be characterized as, but is not limited to, a form in which the hydrophilic amino acid RALA is bound to the amphiphilic polymer polyetherimide (PEI).

[0031] In the present invention, D may be characterized by including a peptide sequence having a cell-penetrating function. The peptide having a cell-penetrating function is not particularly limited as long as it has the characteristic of entering cells through a mechanism of endocytosis, but is preferably selected from the group consisting of cell-penetrating peptides disclosed in Korean Patent Registration No. 10-0951719 or variants thereof.

[0032] In one embodiment of the present invention, D may be a peptide selected from the group consisting of 4 to 12 arginines, 4 to 12 lysines, and 2 cysteines, or a combination thereof, but is not limited thereto. Peptides or peptide analogs other than the above-mentioned peptides may also be used as long as they can permeate the cell membrane.

[0033] In the present invention, the above-mentioned E can be characterized by including a peptide sequence that recognizes the surface of a B cell or a T cell.

[0034] The above-mentioned B cells or T cells refer to cells targeted by the mRNA that is to be delivered into the cells by binding to the above-mentioned RNA-binding peptide.

[0035] In the present invention, the above E is a sequence that binds to the surface of existing B cells or T cells, and can be newly discovered and applied through phage display technology.

[0036] In one embodiment of the present invention, E may be, but is not limited to, a peptide represented by the amino acid sequence of SEQ ID NO: 17 that targets B cells and / or T cells. SEQ ID NO: 17: HTHGAARVPDHR

[0037] In the present invention, the peptide C that promotes endosomal escape can be additionally included between the above B and D.

[0038] The C may be characterized by including a peptide sequence that promotes endosomal escape. It is not particularly limited as long as it promotes endosomal escape of the conjugate, but preferably can be selected from the group consisting of endosome-degrading peptides or variants thereof disclosed in US 2020 / 0207834 A1.

[0039] In one embodiment of the present invention, C may be characterized as a peptide consisting of 4 to 12 histidines, but is not limited thereto.

[0040] In the present invention, ABDE or ABCDE of the above structural formula 1 can be characterized by being produced by chemical synthesis or recombinant expression.

[0041] In another aspect, the present invention relates to nanoparticles to which the peptide-based conjugate and mRNA are bound.

[0042] In the present invention, the mRNA can be characterized by encoding a target protein, recombinant protein, or viral antigen whose expression is to be increased. It can be mRNA with a natural structure having a 3' cap and a 5' poly A tail, or it can be mRNA with a sulfhydryl group or amine group attached to the 5' end, followed by chemical crosslinking with the sulfhydryl group of cysteine ​​in the cell permeation and B / T cell recognition peptide.

[0043] In one embodiment of the present invention, the expression of mutant KRAS was suppressed using mRNA for mutant KRAS. The mRNA sequence encoding the protein that degrades KRAS may be, but is not limited to, mRNA having the nucleotide sequences of SEQ ID NO: 18 and SEQ ID NO: 19. SEQ ID NO: 18: mRNA sequence 1 encoding a protein that degrades KRAS 5' cap-5'UTR- SEQ ID NO: 19: mRNA sequence 2 encoding a protein that degrades KRAS 5' cap-5'UTR-

[0044] In the present invention, after preparing the above structural formula 1, mRNA is bound to structural formula 1, specifically to A in structural formula 1, and when left for a certain period of time, nanoparticles are formed by self-assembly.

[0045] The nanoparticles can be characterized by a size of 10 to 200 nm, but are not limited to this.

[0046] In the present invention, the ratio of the molecular weight of the mRNA to the molecular weight of the conjugate can be characterized as being 1:1 to 1:100.

[0047] In the present invention, the above mRNA can be characterized by being used in an mRNA vaccine, but is not limited thereto.

[0048] In another aspect, the present invention relates to a composition for delivering mRNA into B cells or T cells, comprising nanoparticles to which the peptide-based conjugate is bound.

[0049] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a viral infection, comprising nanoparticles to which the peptide-based conjugate and mRNA are bound.

[0050] The viral infection can be, but is not limited to, a cold, influenza, infectious mononucleosis, cytomegalovirus infection, measles, polio, yellow fever, dengue fever, hepatitis B, hepatitis C, AIDS, or COVID-19.

[0051] In the present invention, the term "prevention" means any action of suppressing or delaying a disease by administering the composition, and the term "treatment" means any action of improving or curing the symptoms of a disease by administering the composition.

[0052] In the present invention, the pharmaceutical composition may be formulated in any dosage form selected from the group consisting of injections, oral preparations, patches, liquids, capsules, granules, tablets, powders, sprays, ointments, gels, mucosal preparations, and suppositories, but is not limited thereto. These preparations may be prepared by conventional methods used in the art or by methods disclosed in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton PA, and may be formulated in various dosage forms depending on the disease or ingredient. However, the above descriptions are merely illustrative, and the formulations applicable to the present invention are not limited thereto.

[0053] In the present invention, the pharmaceutical composition may further comprise an acceptable auxiliary agent, such as a carrier. Pharmaceutically acceptable carriers include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to the composition to formulate it into an injectable dosage form, such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets. However, the above descriptions are merely illustrative, and the auxiliary agents and carriers usable in the present invention are not limited to these.

[0054] In still another aspect, the present invention relates to a method for preventing or treating a viral infection, which comprises administering the above-mentioned nanoparticles.

[0055] In still another aspect, the present invention relates to the use of the nanoparticles for the prevention or treatment of viral infections.

[0056] In still another aspect, the present invention relates to use of the above nanoparticles for producing a drug for preventing or treating a viral infection.

[0057] In the present invention, the above-mentioned methods, uses and applications include the nanoparticles according to the present invention and relate to pharmaceutical compositions containing the nanoparticles, and the description of the overlapping content with the above-mentioned nanoparticles and pharmaceutical compositions will be omitted. [Example]

[0058] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0059] Example 1. Synthesis of transmitter peptides Amino acids and reagents required for synthesis were purchased from GL biochem and Sigma-Aldrich. Peptides were synthesized from the C-terminus using a peptide synthesizer using F-moc solid-state chemical synthesis.

[0060] That is, synthesis was performed using Rink resin (0.075mmol / g, 100-200mesh, 1% DVB crosslinking) to which Fmoc-(9-Fluorenylmethoxycarbonyl) was bound as a blocking group. 50mg of Rink resin was placed in a synthesizer, and the resin was swelled with DMF. 20% piperidine / DMF solution was then used to remove the Fmoc group.

[0061] Starting from the C-terminus, 5, 10, and 5 equivalents of 0.5 M amino acid solution (solvent: dimethylpormamide, DMF), 1.0 M DIPEA (solvent: dimethylpormamide & enmethylpyrrolidone, DMF & NMP), and 0.5 M HBTU (solvent: dimethylpormamide, DMF) were added in the order of the sequence, and the mixture was reacted under a nitrogen stream for 1 to 2 hours.

[0062] After each deprotection and coupling step, the resin was washed twice with DMF and isopropanol. After coupling the final amino acid, deprotection was also performed to remove the Fmoc group. After the reaction was completed, the resin was washed with DMF and MeOH and dried in a vacuum oven.

[0063] Trifluoroacetic acid (TFA) cleavage cocktail was added at a ratio of 20 mL per 1 g of resin and shaken for 3 hours, after which the resin and the peptide-dissolved cocktail were separated by filtering. An excess amount of cold ether was added directly to the filtered TFA cocktail solution containing the dissolved peptide to crystallize the peptide into a solid phase, which was then separated by centrifugation. The TFA cocktail was then completely removed by several washes with ether and centrifugation processes. The peptide thus obtained was dissolved in distilled water and lyophilized.

[0064] After freeze-drying, the product was separated and purified by high performance liquid chromatography (Shimadzu, Japan). 18 The column was analyzed by flowing 0.1% TFA / H2O and 0.092% TFA / acetonitrile at a flow rate of 1 mL / min for 30 minutes, varying the concentration from 0 to 60%, with the UV detector wavelength set to 220 nm.

[0065] The purification was carried out using a 2.2 cm diameter column at a flow rate of 20 mL / min under the same solvent and detection wavelength conditions. The molecular weight of the purified peptide was confirmed by mass spectrometry.

[0066] Example 2. Preparation of nanoparticles by binding of carrier peptide to mRNA The carrier peptide prepared in Example 1 was bound to mRNA of a gene encoding a His tag protein or an eGFP (enhanced green fluorescent protein) protein to prepare nanoparticles.

[0067] Five mg of the carrier peptide was dissolved in 1 mL of nuclease-free water (Invitrogen, AM9938). One mg of mRNA was dissolved in 1 mL of nuclease-free water (Invitrogen, AM9938). The carrier peptide solution and mRNA solution were mixed slowly in small amounts at N / P (nitrogen / phosphate) ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, and 20:1.

[0068] The formed nanoparticles were confirmed through a gel retardation test. Electrophoresis was performed by loading mRNA and carrier peptide at ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, and 20:1 onto a 2% (w / v) agarose gel, with an amount of mRNA equivalent to 0.1 μg. Single mRNA was observed to move downwards, which is a positive charge (Figure 3), but when nanoparticles were formed, they did not move downwards from a 10:1 ratio. This means that the carrier peptide neutralized the particle surface.

[0069] Furthermore, when the nanoparticles were dissociated with 0.05% SDS, it was confirmed that the mRNA returned to the gel (Figure 3). Furthermore, using a transmission electron microscope (TEM, JEM-1230, JEOL), nanoparticles composed of a conjugate of mRNA and carrier peptide were confirmed.

[0070] The particle size and zeta potential were measured using an electrophoretic light scattering spectrophotometer (ELS Z-1000, Otsuka Portal, Japan) (Figure 3). The nanoparticles produced were confirmed to have a diameter of 100–150 nm.

[0071] Example 3. Confirmation of delivery and expression of nanoparticles into cells The nanoparticles prepared in Example 2 were administered to the culture medium of Raw264.7 cells to confirm their intracellular expression. Nanoparticles were formed with a carrier peptide using mRNA expressing eGFP, and the nanoparticles were delivered into the cells and eGFP protein was expressed using a confocal microscope (Figure 4).

[0072] To test the cell penetration ability of nanoparticles containing a conjugate of mRNA and a carrier peptide, nanoparticles were prepared in the same manner as in Example 2. Raw264.7 (mouse macrophage cells, TIB-71, ATCC) were added to each 1x10 4 After 24 hours, 20 μg of mRNA-transmitter peptide conjugate (mRNA amount) was added to the culture medium for 24 hours. The cells were then fixed with 10% neutral formalin solution and analyzed using a confocal parallax scanning microscope.

[0073] As a result, a significantly increased amount was observed in the cytoplasm of Raw264.7 cells (Figure 4). Therefore, it was confirmed that nanoparticles containing mRNA-loaded carrier peptide conjugates can effectively penetrate cells.

[0074] Example 4. Confirmation of expression of KRAS-degrading protein and reduction of KRAS by mRNA delivered into cells In addition, the amount of protein expressed in cells by nanoparticles composed of mRNA and carrier peptide was confirmed by Western blot (Figure 5).

[0075] H358 cells were seeded into 6-well plates at 60% density and then starved overnight in serum-free RPMI-1640 medium 48 hours later. Cells were treated for 24 hours with medium containing 20 μg (mRNA amount) of nanoparticles containing mRNA and carrier peptide conjugates. As a control, 20 μg of mRNA mixed with lipopectin was used. Proteins were lysed using RIPA lysis buffer (25 mM Tris·HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors. Proteins were quantified using a BCA protein assay (23227, Thermo Scientific), and His-tagged protein expression was confirmed by Western blot. For Western blots, equal amounts of samples were loaded onto a 12% SDS PAGE gel along with a size marker and electrophoresed for approximately 2 hours, then transferred to a nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and incubated overnight with a primary antibody (His tag, Abcam, ab18184) at a 1:1000 ratio. The membrane was then washed with TBST containing 0.1% Tween-20 and incubated with an HRP-conjugated secondary antibody (anti-Mouse, A120-101P, BETHYL Lab.) for 1 hour, followed by chemiluminescence detection using ECL substrate.

[0076] As a result, as shown in Figure 5, when nanoparticles composed of the peptide-lipid conjugate of the present invention were used, the amount of protein expressed by mRNA in cells increased with a higher N / P ratio than when lipid nanoparticles such as lipofectamine were used.

[0077] To confirm the expression of GTP-bound KRAS (active KRAS), cell lysates were incubated with beads containing RBD-RAF-GST (RAS binding domain RAF GST) for 1 hour. After washing the beads three times with lysis buffer, proteins were dissociated from the beads with 2X loading buffer. For Western blots, equal amounts of samples were loaded onto a 12% SDS-PAGE gel along with a size marker and electrophoresed for approximately 2 hours before transferring to a nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and incubated overnight with primary antibodies (GST (Abcam, ab19256) and KRAS (Santa Cruz, sc-30)) at a 1:1000 ratio. The membrane was then washed with TBST containing 0.1% Tween-20 and incubated with HRP-conjugated secondary antibodies (anti-rabbit, A90-116P, BETHYL Lab.; anti-mouse, A20-101P, BETHYL Lab.) for 1 hour, after which chemiluminescence was confirmed using ECL substrate.

[0078] As a result, as shown in Figure 5, it was confirmed that the protein was expressed and the expression of active KRAS was significantly reduced in the nanoparticles.

[0079] Example 5. Confirmation of targeting ability of B cell / T cell target peptide in B cell / T cell The B cell / T cell targeting peptide (BTBP, SEQ ID NO: 17) was labeled with Cy5.5 and treated with B cells (Primary CD19+ B cells, PCS-800-018, ATCC) and T cells (Primary CD8+ Cytotoxic T cells, PCS-800-017, ATCC) at 100 μM for 1 hour. Cells were then fixed with 4% neutral formalin solution and analyzed using a confocal differential scanning microscope (LSM980 with Airyscan2, Carl Zeiss). Significant increases in fluorescence intensity were observed at the plasma membrane of both B and T cells (Figure 6). Therefore, we confirmed that BTBP effectively targets both B and T cells.

[0080] The cells were then washed three times with DPBS containing 5% FBS and analyzed for binding to Cy5.5-labeled BTBP using flow cytometry (Figure 7). As a result, it was confirmed that the peptide of SEQ ID NO: 17 binds to B cells and T cells.

[0081] Example 6. Nanoparticle fabrication The COVID-19 vaccine mRNA and RNA-binding peptide (SEQ ID NO: 1 or 3) were mixed at a volume ratio of 1:1, and the cartridge was connected to the Nanoassembly Ignite (Precision Nanosystems). The reaction proceeded at 1.3 mL / min. After primary complex formation, the mixture was incubated at room temperature for 30 minutes.

[0082] Then, 2 mL of the primary complex sample and 1 mL of the amphiphilic polypeptide (SEQ ID NO: 16) were reacted in a volume ratio of 2:1, with a final volume of 3 mL and at 1.3 mL / min, by connecting the cartridge to Nanoassembly Ignite.

[0083] After the secondary complex formation, the mixture was incubated at room temperature for 30 minutes. Then, 3 mL of the sample obtained by the secondary complex formation and 1 mL of the B cell / T cell binding peptide (SEQ ID NO: 17) were connected to the cartridge of the Nanoassembly Ignite, and the reaction was carried out at a volume ratio of 3:1, a final volume of 4 mL, and a flow rate of 1.3 mL / min.

[0084] Finally, the final sample was filtered using a cellulose acetate filter (0.2 μm) and then concentrated with nitrogen gas to a volume of 200 μL. The weight ratios of the nanoparticle synthesis components, mRNA: RNA peptide-binding peptide: amphiphilic polypeptide: B cell / T cell binding peptide, were prepared as follows: Sample 1 = 1:2:2:5 Sample 2 = 1:3:3:5

[0085] The control LNPs were prepared by mixing an organic phase and an aqueous phase at a fixed weight ratio relative to the mRNA. The organic phase was prepared by mixing an ionic lipid (SM-102, Moderna), a phospholipid (DSPC; Avanti, 816-94-4), cholesterol (Cholesterol; Avanti 700100P), and a PEG lipid (DMG-PEG; Avanti 880151P) in ethanol (Sigma E7023) at a ratio of ionic lipid:phospholipid:cholesterol:PEG lipid = 50 (6.25 mM):10 (1.25 mM):38.5 (4.8125 mM):1.5 (1.875 mM).

[0086] The aqueous phase was prepared by mixing mRNA with citrate (Sigma 854) and DPBS (Cytiva SH30028.02). The organic and aqueous phases were mixed at a volume ratio of 1:3 and mixed at a flow rate of 12 mL / min to prepare LNPs. The prepared LNPs were dialyzed against PBS for 16 hours using a dialysis cassette (Merk, UFC901024) to remove ethanol and to match the pH of the LNPs to the body's pH. The size and shape of the prepared nanoparticles were observed using a TEM (JEM-1400, JEOL) (Figure 8). Samples 1 and 2 all showed a size distribution of 129–155 nm and were spherical.

[0087] Example 7. Evaluation of antibody formation against SARS-CoV-2 Spike Glycoprotein S1 using ELISA The nanoparticles prepared in Example 6 were injected intramuscularly into the thigh of C57BL / 6 mice, and the mice were sacrificed two weeks later. The collected blood was centrifuged to separate the serum.

[0088] Recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein S1 (ab275927) was added to the wells at 100ng / 50μL using ELISA coating buffer and coated for 18 hours at 4℃. The coating buffer was then removed from the plate, and the plate was washed twice with washing buffer (PBST, PBS + 0.05% Tween 20). 100μL / well of PBS + 1% BSA buffer was added and incubated at 37℃ for 2 hours to block the reaction.

[0089] After washing twice with wash buffer, the separated serum was diluted with PBS + 1% BSA buffer and serially diluted starting from 1:40 for each individual. 100 μL of the diluted serum was added to each well and incubated at 37°C for 1 hour. After washing three times with wash buffer, Horesradish peroxidase (HRP)-conjugated secondary antibody was diluted 1:5000 and added to each well at 50 μL for 1 hour at 37°C. After washing five times with wash buffer, 50 μL of TMB solution was added to each well and incubated at RT for 10 minutes. 50 μL of stop solution (2M H2SO4) was then added to each well to terminate the reaction. Measurements were taken at 450 nm using a microplate reader (Figure 9).

[0090] As a result, ELISA analysis confirmed that antibodies against SARS-CoV-2 Spike Glycoprotein S1 were formed in the experimental groups injected with Samples 1 and 2. This proves that the injected nanoparticles deliver COVID-19 vaccine mRNA into the body, effectively expressing SARS-CoV-2 Spike Glycoprotein S1 and inducing an immune response.

[0091] Example 8. Evaluation of immune response using ELISPOT The nanoparticles prepared in Example 6 were injected intramuscularly into the thigh of C57BL / 6 mice, which were sacrificed two weeks later. The spleens were removed from the mice and grated using a 40 μm pore strainer in 5 mL of 1X HBSS media. The spleens were then transferred to a 50 mL conical tube and centrifuged at 2000 rpm for 5 minutes. After removing the medium, 5 mL of ACK buffer was added and incubated at room temperature for 5 minutes to remove red blood cells, yielding splenocytes.

[0092] Spleen cells obtained from the spleens of mice immunized with the candidate substance were dispensed onto an IFN-γ antibody-coated microplate, and then cell culture medium was added as a negative control, a cell stimulation cocktail as a positive control, and SARS-CoV-2 spike peptide (Genscript RP30020 SARS-CoV-2 Spike Glycoprotein-crude, 2 vials (25■ / peptide)) was added as an experimental group, and the cells were cultured at 37°C in a CO2 incubator for 18 hours.

[0093] Biotinylated IFN-γ monoclonal antibody was added to each well and incubated at room temperature for 1 hour. Streptavidin-alkaline phosphatase was then added to each well and incubated at room temperature for 1 hour. 5-bromo-4-chloro-3-indolyl-phosphate / nitro blue tetrazolium (BCIP / NBT) solution was added and incubated at room temperature for 10 minutes. After washing, the colored spots were counted and analyzed (Figure 10).

[0094] As a result, it was confirmed that the SARS-CoV-2 spike peptide induced an immune response in spleen cells isolated from mice injected with Sample 1 and Sample 2. Therefore, it was proven that the injected nanoparticles delivered COVID-19 vaccine mRNA into the body, effectively expressing SARS-CoV-2 Spike Glycoprotein S1 and inducing an immune response.

[0095] Example 9. Evaluation of antibody formation against human ACE2 using hACE2 immunoassay The nanoparticles prepared in Example 6 were injected intramuscularly into the thigh of C57BL / 6 mice, and the mice were sacrificed two weeks later. The collected blood was centrifuged to separate the serum. After serum separation, the nanoparticles were heat inactivated at 56°C for 30 minutes. This experiment was carried out using Lumit® (Promega). TM This was performed using the SARS-CoV-2 Spike RBD:hACE2 immunoassay kit.

[0096] First, serum was diluted in 1X immunoassay reaction buffer to prepare a 10X sample mix, and 5 μL was dispensed into a 96-well white plate. Then, 10 μL of rFC-RBD Reagent (III.B.2) was added to each well. Next, 10 μL of mFC-ACE2 Reagent (III.B.2) was added. Next, Lumit (III.B.3) was added. TM 25 μL of Antibody Mix reagent was added to all wells, and the reagent was mixed on a plate shaker at room temperature for 2 minutes and then incubated for 60 minutes. TM After adding the detection reagent, the mixture was incubated at room temperature for 30 minutes and then measured using a plate-readable luminescence system (Figure 11).

[0097] As a result, it was confirmed that antibodies against human ACE2 were formed in the serum of mice injected with Samples 1 and 2. Therefore, it was proven that the injected nanoparticles delivered COVID-19 vaccine mRNA into the body, effectively expressing SARS-CoV-2 Spike Glycoprotein S1 and generating an immune response. [Industrial Applicability]

[0098] The nanoparticles composed of peptide-based conjugates to which mRNA is bound according to the present invention can effectively deliver mRNA into cells and can be used as vaccines to prevent infectious diseases using mRNA. Compared to existing lipid nanoparticles, the expression efficiency and safety are improved, thereby enhancing the vaccine effect of mRNA.

[0099] While the present invention has been described in detail above, it is obvious to those skilled in the art that the specific details are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

[0100] Sequence Catalog Free Text Electronic file attached.

Claims

1. A peptide-based conjugate having the following structural formula 1 as a base unit: [Structural formula 1] A-B-D-E where A is an RNA-binding peptide, B is an amphiphilic polypeptide or amphiphilic polymer; D is a peptide with cell-penetrating function, E is a peptide that recognizes the surface of B cells or T cells.

2. The conjugate according to claim 1, wherein A is a peptide represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 15.

3. The conjugate of claim 1, wherein D is a peptide selected from the group consisting of 4 to 12 arginines, 4 to 12 lysines, and 2 cysteines, or a combination thereof.

4. The conjugate according to claim 1, wherein E is a peptide represented by the amino acid sequence of SEQ ID NO:

17.

5. The conjugate of claim 1, further comprising C, a peptide that promotes endosomal escape, between B and D.

6. The conjugate according to claim 5, wherein C is a peptide consisting of 4 to 12 histidines.

7. The conjugate according to claim 5, wherein the ABDE or ABCDE is produced by chemical synthesis or recombinant expression.

8. A nanoparticle comprising the peptide-based conjugate of any one of claims 1 to 7 and mRNA bound thereto.

9. The nanoparticle of claim 8, wherein the mRNA encodes a target protein, a recombinant protein, or a viral antigen whose expression is to be increased.

10. The nanoparticle of claim 8, wherein the mRNA is bound to A in Formula 1 and then formed through self-assembly of a peptide-based conjugate.

11. Nanoparticles according to claim 10, characterized in that they have a size of 10 to 200 nm.

12. The nanoparticles according to claim 8, wherein the molecular weight ratio of the mRNA to the conjugate is 1:1 to 1:

100.

13. A composition for delivering mRNA into B cells or T cells, comprising the nanoparticles according to claim 8.

14. A pharmaceutical composition for preventing or treating a viral infection, comprising the nanoparticles according to claim 8.

15. The pharmaceutical composition according to claim 14, wherein the viral infection is a cold, influenza, infectious mononucleosis, cytomegalovirus infection, measles, polio, yellow fever, dengue fever, hepatitis B, hepatitis C, AIDS, or COVID-19.

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