Cationic amphiphilic polypeptide for RNA delivery and manufacturing method thereof

Abstract

The present invention relates to a cationic amphiphilic polypeptide for RNA delivery and a method for manufacturing same. It was confirmed that a polypeptide polymer in which a conjugate having diethylenetriamine (DET) bonded to aspartic acid and a conjugate having octylamine (OCT) bonded to aspartic acid are bound in a specific sequence exhibits superior mRNA transcription efficiency compared to polymers in which the conjugates are bound in different orders. Thus, the polypeptide polymer, a manufacturing method therefor, and an RNA delivery composition containing same can be advantageously utilized.

Description

Cationic amphiphilic polypeptide for RNA delivery and method for producing the same The present invention relates to a cationic amphiphilic polypeptide for RNA delivery and a method for producing the same. In vitro transcribed (IVT) mRNA has been progressively developed for use in vaccination, cancer immunotherapy, and genome editing. The success of IVT mRNA therapeutics is hampered by the fragility and immunogenicity of single-stranded RNA molecules, as well as the inefficient cellular uptake of nucleic acids due to their negatively charged macromolecular structure. Synthetic carriers, such as lipid nanoparticles and polymeric nanoparticles (polyplexes), have been developed to protect mRNA from nucleases and enhance internalization into target cells. Recently, cationic polymers have been developed that contain amino and hydrophobic groups in random or defined arrangements. The amino groups are responsible for electrostatic associations between the cationic polymer and the anionic payload, as well as for endosomal escape of the nanoparticles after cellular internalization into target cells / tissues. The hydrophobic groups allow for hydrophobic interactions during nanoparticle formation, which increases the colloidal stability of polymeric nanoparticles in aqueous solutions, both in vitro and in vivo. The behavior of polymers in solution is highly dependent on the monomer sequence and its length. A well-defined polymer sequence determines the higher-order structure that leads to the desired functionality. Therefore, sequence-controlled polymers, in which monomer units of different chemical properties are arranged in an ordered manner, can more effectively control the structure-property relationships within polymer materials. The quantitative structure-property relationships of sequence-controlled polymers have been gradually understood, including the phase separation of oppositely charged polyelectrolytes into complex coacervates. For example, the sequence of charged polyelectrolytes influences the strength of the electrostatic interaction between two molecules, leading to phase separation and coacervate formation. Sequence-controlled polymers exhibit unique thermal and pH responses in aqueous solution that are distinct from random copolymers. Recently, various polymerization methods, such as radical polymerization and supramolecular polymerization, are being researched and developed to synthesize sequence-controlled polymers. The purpose of the present invention is to provide a polypeptide polymer represented by the following chemical formula 1. [Chemical Formula 1] In the above chemical formula 1, n is one of 1 to 20, a and b may be the same or different and are one of 1 to 5, c and d may be the same or different and are one of 1 to 20, and R 1 and R 2 may be the same or different, and are hydrogen or (C4-C12)cycloalkyl. Another object of the present invention is to provide a composition for RNA delivery comprising the polypeptide polymer. Another object of the present invention is to provide a reagent composition for promoting RNA transcription comprising the polypeptide polymer. Another object of the present invention is to provide a method for producing a polypeptide polymer represented by the chemical formula 1, comprising the steps of: a first step for producing a conjugate in which diethylenetriamine (DET) is bound to aspartic acid; a second step for producing a conjugate in which octylamine (OCT) is bound to aspartic acid; and a third step for combining the conjugate of the first step and the conjugate of the second step. To achieve the above purpose, the present invention provides a polypeptide polymer represented by the following chemical formula 1. [Chemical Formula 1] In the above chemical formula 1, n is one of 1 to 20, a and b may be the same or different and are one of 1 to 5, c and d may be the same or different and are one of 1 to 20, and R 1 and R 2 may be the same or different, and are hydrogen or (C4-C12)cycloalkyl. In addition, the present invention provides a composition for RNA delivery comprising the polypeptide polymer. In addition, the present invention provides a reagent composition for promoting RNA transcription comprising the polypeptide polymer. In addition, the present invention provides a method for producing a polypeptide polymer represented by Chemical Formula 1, comprising the steps of: a first step of producing a conjugate in which diethylenetriamine (DET) is combined with aspartic acid; a second step of producing a conjugate in which octylamine (OCT) is combined with aspartic acid; and a third step of combining the conjugate of the first step and the conjugate of the second step. According to the present invention, it has been confirmed that a polypeptide polymer in which a conjugate of aspartic acid and diethylenetriamine (DET) and a conjugate of aspartic acid and octylamine (OCT) are combined in a specific order has superior mRNA transcription efficiency compared to a polymer in which the conjugates are combined in a different order, and thus the polypeptide polymer, a method for producing the same, or an RNA delivery composition comprising the same can be usefully utilized. Figure 1 shows a synthetic pathway for a sequence-controlled polypeptide having Fmoc-L-Asp(OCT)-OH (Figure 1A); and diethylenetriamine (hereinafter referred to as DET) and octylamine (hereinafter referred to as OCT) moieties. Figure 2 shows the NMR analysis results of Fmoc-L-Asp(OCT)-OBn (Experimental Example 2-2-3) (the upper drawing is 1 H NMR, bottom figure is 13 C NMR data). Figure 3 shows the NMR analysis results of Fmoc-L-Asp(OCT)-OH (Experimental Example 2-2-4) (the upper drawing is 1 H NMR, bottom figure is 13 C NMR data). Figure 4 shows the results of predicted and measured mass spectra analysis of polypeptides. A; DODODODO, B; DDOODDOO, C; DDDDOOOO, D; 12mer DDOO (=DDOODDOODDOO), E; 16mer DDOO (=DDOODDOODDOODDOO), F; 20mer DDOO (=DDOODDOODDOODDOODDOO). Figure 5 shows the HPLC spectrum analysis results of polypeptides. A; DODODODO, B; DDOODDOO, C; 12mer DDOO, D; 16mer DDOO, E; 20mer DDOO. Figure 6 shows the results of HPLC spectrum (left) and mass spectrum (right) analysis of polypeptides. A; 20-mer DDO(O), B; 20-mer (D)DOO, (C) 20-mer (D)(D)OO, and D; 20-mer (D)D(O)O. In the above experimental groups, D or O in parentheses is the D-form, and D or O not in parentheses is the L-form. Parentheses are used only to indicate the D-form. Figure 7 shows the structure and citation of a polypeptide manufactured in the present invention. Figure 8 shows the results of D / L isomer structure analysis of DDOODDOO measured through GC-MS. Figure 9A shows the results of analyzing the degree of protonation of an octameric polypeptide (pKa graph); Figure 9B shows the results of analyzing the light scattering intensity of an octameric polypeptide; Figure 9C shows the results of analyzing the light scattering intensity of 12-, 16-, and 20-mer DDOO polypeptides; Figure 9D shows the results of analyzing the circular dichroism (CD) spectra of an octameric polypeptide and a 20-mer DDOO polypeptide; and Figure 9E shows the results of analyzing the molecular structure of a DDOODDOO polypeptide. Figure 10 shows the structures of DDDDOOOO (Figure 10A), DODODODO (Figure 10B), and DDDDDDDD (Figure 10C) predicted through all-atom molecular dynamics simulations (AAMD). The backbone is shown in red, the DET group in blue, the OCT group in gray, and the B-turn-like structure in purple. Figure 11 shows the results of agarose gel electrophoresis analysis of polyplexes prepared according to the N / P ratio between the sequence-controlled polypeptide and FLuc-mRNA. The N / P ratio is defined as the molar ratio of the amino group in the DET unit to the phosphate group in the mRNA. Figure 12 shows the results of luminescence intensity analysis of A549 cells transfected with FLuc-mRNA for 24 hours. Specifically, Figure 12A shows the results of luminescence intensity analysis of cells transfected with polyplexes loaded with mRNA prepared with octameric polypeptides at various N / P ratios; Figure 12B shows the results of luminescence intensity analysis of cells transfected with polyplexes transfected with 12-, 16-, and 20-mer DDOO (N / P=5) polypeptides; Figure 12C shows the results of cellular uptake analysis of Cy5-mRNA-loaded polyplexes (N / P=5,500 ng mRNA / well) in the cells (Figure 12B); and Figure 12D shows the results of agarose gel electrophoresis analysis of IVT mRNA of polyplexes (N / P=5) cultured in 10% FBS at 37°C for 1 hour; And Figure 12E shows the results of luminescence intensity analysis of cells transfected with FLuc-mRNA at 100 ng mRNA / well for 24 hours. All results are expressed as mean ± standard deviation (n = 4). Figure 13 is the original data of Figure 12C, which shows the results of cellular uptake analysis of Cy5-mRNA-loaded polyplexes (N / P = 5,500 ng mRNA / well) in A549 cells cultured for 4 hours. The control group (non-polyplex-treated group) is shown in blue (★), and the polyplex-treated group is shown in red (☆). Figure 14 shows the results of analyzing the in vitro gene editing efficacy using HEK293-loxP-GFP-RFP (Neo) cells that exhibit red fluorescence after LoxPs are cleaved by Cre recombinase. Specifically, Figure 14A is a schematic diagram of HEK293 cells expressing the LoxP-GFP-stop-LoxP-RFP cassette, Figure 14B is a CLSM observation image of HEK293-loxP-GFP-RFP treated with 20mer DD00 / Cre-mRNA for 48 hours (100 ng mRNA / well), and Figure 14C is the results of analyzing Cre recombinase activity through the number of GFP or / and RFP expressing cells and their fluorescence intensity. Figure 15 shows the results of an in vitro gene editing assay using HEK293-loxP-GFP-RFP (Neo) cells that exhibit red fluorescence after LoxPs are cleaved by Cre recombinase. Specifically, Figure 15A shows cells that were not treated with the sample, Figure 15B shows cells treated with 20-mer DDOO and Cre-mRNA for 48 hours, and Figure 15C shows cells treated with Lipofectamine 3000 (positive control) for 48 hours. Figures 16A to 16D show the results of ITC (isothermal titration calorimetry) profile analysis of 8mer DDOO / ssRNA (Figure 16A), 12mer DDOO / ssRNA (Figure 16B), 16mer DDOO / ssRNA (Figure 16C), and 20mer DDOO / ssRNA (Figure 16D). The upper figures in Figures 16A to 16D are raw ITC data, and the lower figures show a plot of heat flow according to the molar ratio between the polypeptide and ssRNA. Figure 16E shows the results of AAMD simulation analysis between the DDOO polymer and 20mer RNA, and Figure 16F is an optical image of a polyplex containing 20nt ssRNA (N / P=5). Hereinafter, the present invention will be described in more detail. The present invention provides a polypeptide polymer represented by the following chemical formula 1. [Chemical Formula 1] In the above chemical formula 1, n is one of 1 to 20, a and b may be the same or different and are one of 1 to 5, c and d may be the same or different and are one of 1 to 20, and R 1 and R 2 may be the same or different, and may be hydrogen or (C4-C12)cycloalkyl, Preferably, n is one of 1 to 5, a and b may be the same or different and are one of 1 to 3, c and d may be the same or different and are one of 5 to 10, and R 1 and R 2 may be the same or different, and may be hydrogen or (C4-C8)cycloalkyl, More preferably, n is one of 1 to 5, a and b are one of 1 to 3, c and d are one of 5 to 10, and R 1 and R 2 may be hydrogen or (C4-C8)cycloalkyl. The above polymer may be in a form in which diethylenetriamine (DET) or octylamine (OCT) is bound to aspartic acid, and the form in which diethylenetriamine (DET) or octylamine (OCT) is bound to aspartic acid may be in the form of L-form or D-form. The above polypeptide polymer can increase RNA transcription efficiency, and the RNA can be one or more selected from the group consisting of mRNA, siRNA, ASO (Antisense oligonucleotide), and gRNA (guide RNA), but is not limited thereto. The above mRNA may be an in vitro transcribed (IVT) mRNA. In addition, the present invention provides a composition for RNA delivery comprising the polypeptide polymer. In addition, the present invention provides a reagent composition for promoting RNA transcription comprising the polypeptide polymer. In addition, the present invention provides a method for producing a polypeptide polymer represented by Chemical Formula 1, comprising the steps of: a first step of producing a conjugate in which diethylenetriamine (DET) is combined with aspartic acid; a second step of producing a conjugate in which octylamine (OCT) is combined with aspartic acid; and a third step of combining the conjugate of the first step and the conjugate of the second step. Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art. [Experimental Example 1] Experimental Preparation Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin-EDTA, and ultrapure agarose were purchased from ThermoFisher Scientific (Waltham, MA, USA). HEPES buffer (1 M, pH 7.3) was purchased from Amresco (Solon, OH, USA). A549 human lung cancer cells were purchased from ATCC (Manassas, VA, USA). HEK293-loxP-GFP-RFP (Neo) cells were purchased from GenTarget Inc. (Manassas, VA, USA). FLUC-mRNA (Firefly luciferase-coded mRNAs L-7202) and Cre-mRNA (Cre recombinase-coded mRNAs, L-7211) were purchased from TriLink Biotechnologies (San Diego, CA, USA). Organic solvents and reagents were purchased and used without further purification unless otherwise stated. Single-stranded RNA (ssRNA) (5'-AUGA GGACG CCAAG AACAU-3') was synthesized by Macrogen (Seoul, Korea). Fluorescently labeled FLUC-mRNA was prepared by attaching a fluorescent dye using the Label IT Cy5 labeling kit (Mirus Bio Corporation, Madison, WI, USA). Melting points (mp) were measured using a Leica Galen III microscope and expressed in degrees Celsius (℃). Infrared (IR) spectra were recorded using Nicole IR100 with NaCl crystals as film or Nico IR100 with Nujol as solvent. 1 H and 13C NMR spectra were recorded at room temperature using a Bruker Advance NEO 400 MHz with a Prodigy CPBBBO BB-H&F z-gradient cryo-probe spectrometer. Chemical shifts were expressed in ppm using reported shifts and relative to solvent signals (1H / 13C: deuterated chloroform CDCl3 7.26 / 77.2 ppm, dimethyl sulfoxide DMSO-d6 2.49 / 39.5 ppm) as internal standards. Coupling constants (J) were expressed in hertz and abbreviated to account for multiplicities as follows: s; singlet, d; doublet, t; triplet, q; quintet, sext; sextet, m; multiplet, br; broad. Mass spectra were recorded using electrospray ionization (ESI) on a Thermo Q Exactive Focus or Waters ZQ 400 spectrometer. [Experimental Example 2] Polypeptide Synthesis 2-1. Crude purification The monomer reaction was monitored by thin layer chromatography (TLC) using pre-coated aluminum-backed plates (0.2 mm silica gel 60 F254, Merck®) and visualized with UV light. Purification of the compound was performed using silica gel column chromatography (Chromagel 60A SdS.CC 70–200 μm) with solvent mixtures of increasing polarity as eluents. 2-2. Fmoc-L-Asp(OCT)-OH synthesis 2-2-1. Manufacturing of Fmoc-L-Asp(OtBu)-OBn Fmoc-L-Asp(OtBu) (20.0 g, 48.6 mmol) was dissolved in 70 mL of dimethylformamide (DMF). Powered KOH (2.72 g, 48.6 mmol) was then added, followed by benzyl chloride (16.8 mL, 145.8 mmol). The solution was stirred under argon atmosphere and at room temperature for 24 h. 300 mL of aqueous 2 M HCl was added, and the precipitate was filtered under vacuum, washed three times with water, and then three times with hexane to obtain Fmoc-L-Asp(OtBu)-OBn (20.0 g, 82%) as a white solid. The spectral characteristics were consistent with those reported in the literature. The chemical formula of the above Fmoc-L-Asp(OtBu)-OBn is as follows, and the IUPAC name is 1-benzyl 4-(tert-butyl)(((9H-fluoren-9-yl)methoxy)carbonyl)-L-aspartate. 2-2-2. Manufacturing of Fmoc-L-Asp(OH)-OBn Fmoc-L-Asp(OtBu)-OBn (10.0 g, 22.4 mmol) was suspended in DCM (dichloromethane, 15 mL), and TFA (trifluoroacetic acid, 15 mL) was added to the mixture. The solution was stirred at room temperature for 8 h and concentrated under reduced pressure. A trace amount of TFA was removed by co-evaporation with DCM (x 3) to yield Fmoc-Asp(OH)-OBN (9.98 g) as a brown oil. The spectral characteristics were consistent with those reported in the literature. The chemical formula of the above Fmoc-L-Asp(OH)-OBn is as follows, and the IUPAC name is (S)-3-((((9H-(fluoren-9-yl)methoxy)carbonyl)amino)-4-(benzyloxy)-4-L-oxobutanoic acid. 2-2-3. Manufacturing of Fmoc-L-Asp(OCT)-OBn Fmoc-L-Asp(OH)-OBn (9.98 g, 22.4 mmol) was dissolved in DCM (100 mL), and the solution was cooled in an ice bath under an argon atmosphere. HATU (Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium, 8.52 g, 22.4 mmol) was then added to the solution. DIEA (N,N-Diisopropylethylamine, 7.8 mL, 44.8 mmol) was also added to the solution. The mixture was stirred at 0°C for 30 min, and a solution of 1-octylamine (3.7 mL, 22.4 mmol) in DCM (30 mL) was added. The solution was stirred at room temperature for 1 h, transferred to a separatory funnel, washed twice with 2 M HCl aqueous solution and 10% aqueous NaHCO3, and dried over anhydrous Na2SO4. The solution was filtered and evaporated under reduced pressure. The crude mixture was purified by silica gel column chromatography using a mixture of DCM-EtOAc (0-15%) as an eluent to obtain Fmoc-L-Asp(OCT)-OBn (11.5 g, 92%) as a white solid. The chemical formula of Fmoc-L-Asp(OCT)-OBn is as follows, and the IUPAC name is benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -Octyl-L-asparaginate [Benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -octyl-L-asparaginate], and the NMR analysis results were as follows. mp: 121-123℃ IR (CDCl3, υ in cm-1): 3299, 3066, 3033, 2923, 2852, 1755, 1684, 1645, 1547, 1450, 1294. 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 7.5 Hz, 2H), 7.53 (d, J = 7.4 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.31-7.19 (m, 7H), 6.20 (d, J = 8.0 Hz, NH), 5.73-5.63 (m, NH), 5.20-5.09 (m, 2H), 4.62 (s, 1H), 4.57 (dd, J = 8.2, 4.1 Hz, 1H), 4.38-4.30 (m, 1H), 4.27-4.20 (m, 1H), 4.14(t, J = 7.1 Hz, 1H), 3.11 (dd, J = 13.0, 6.4 Hz, 1H), 2.85 (dd, J = 15.6, 4.0 Hz, 1H), 2.66 (dd, J = 15.6, 4.0 Hz, 1H), 1.34 (d, J = 18.3 Hz, 2H), 1.20 (s, 10H), 0.83 (t, J = 6.5 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 171.1 (C), 169.7 (C), 156.4 (C), 144.0 (C), 143.8 (C), 141.3 (C), 141.1 (C), 135.5 (C), 128.6 (CH), 128.6 (CH), 128.4 (CH), 128.2 (CH), 127.8 (2CH), 127.6 (2CH), 127.2 (CH), 127.0 (CH), 125.3 (CH), 125.3 (CH), 120.0 (CH), 67.5 (CH2), 67.4 (CH2), 65.3 (CH2), 51.2 (CH), 47.2 (CH), 39.8 (CH2), 37.7 (CH2), 31.9 (CH2), 29.6 (CH2), 29.3 (CH2), 27.0 (CH2), 22.7 (CH2), 14.2 (CH3). HRMS (ESI+): Calculated for C 34 H40N2NaO5+ [M+H]+ 579.2829, found 579.2824. +13.7 (c 0.8, CHCl3). 2-2-4. Preparation of Fmoc-L-Asp(OCT)-OH Fmoc-L-Asp(OCT)-OBn (3.87 g, 8.29 mmol) was dissolved in a mixture of EtOAc (30 mL) and MeOH (10 mL), and the solution was transferred to a pressure vessel filled with 5% Pd / C (112 mg, 0.05 mmol). A hydrogen cylinder was connected to the pressure vessel, and the reaction mixture was stirred at a pressure of 4 Pa ​​of H2 for 18 h. After that, the reaction mixture was diluted with CHCl3 (200 mL) and refluxed until the product dissolved, and the hot solution was filtered through a cotton pad to obtain Fmoc-L-Asp(OCT)-OH (2.2 g, 57%) as a white solid. The chemical formula of Fmoc-L-Asp(OCT)-OH is as follows, and the IUPAC name is benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -Octyl-L-asparagine [Benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -octyl-L-asparagine], and the NMR analysis results were as follows. mp: 174-176℃ IR (nujol, υ in cm -1 ): 3338, 2929, 2852, 1755, 1690, 1599, 1534, 1463, 1372. 1H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 4.39-4.31 (m, 1H), 4.30-4.16 (m, 3H), 3.02 (dd, J = 12.6, 6.5 Hz, 2H), 2.58 (dd, J = 15.0, 5.1 Hz, 1H), 2.58 (dd, J = 15.0, 5.1 Hz, 1H), 1.42-1.30 (m, J = 6.7 Hz, 2H), 1.20 (s, 10H), 0.83 (t, J = 6.7 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 173.1 (C), 168.9 (C), 155.7 (C), 143.8 (2C), 140.7 (2C), 127.6 (2CH), 127.1 (2CH), 125.2 (2CH), 120.1 (2CH), 65.7 (CH2), 51.0 (CH), 46.6 (CH), 38.6 (CH2), 37.3 (CH2), 31.2 (CH2), 30.1 (CH2), 28.7 (CH2), 28.6 (CH2), 26.4 (CH2), 22.1 (CH2), 13.9 (CH3). HRMS (ESI+): Calculated for C 27 H 34 N2NaO5 + [M+Na] + 489.2360, found 489.2359. -14.1 (c 0.9 DMSO) 2-3.Fmoc-D-Asp(OCT)-OH 합성 2-3-1.Fmoc-D-Asp(OtBu)-OBn제조 Fmoc-D-Asp(OtBu)-OBn was prepared using the same method as the Fmoc-L-Asp(OtBu)-OBn preparation method (Experimental Example 2-2-1). The chemical formula of Fmoc-D-Asp(OtBu)-OBn is as follows, and the IUPAC name is 1-benzyl 4-(tert-butyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-D-aspartate. 2-3-2. Manufacturing of Fmoc-D-Asp(OH)-OBn Fmoc-D-Asp(OH)-OBn was prepared using the same method as the Fmoc-L-Asp(OH)-OBn preparation method (Experimental Example 2-2-2). The chemical formula of Fmoc-D-Asp(OH)-OBn is as follows, and the IUPAC name is (S)-3-((((9H-(fluoren-9-yl)methoxy)carbonyl)amino)-4-(benzyloxy)-4-D-oxobutanoic acid. 2-3-3. Manufacturing of Fmoc-D-Asp(OCT)-OBn Fmoc-D-Asp(OCT)-OBn was prepared using the same method as the Fmoc-L-Asp(OCT)-OBn preparation method (Experimental Example 2-2-3). The chemical formula of Fmoc-D-Asp(OCT)-OBn is as follows, and the IUPAC name is Benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -Octyl-D-asparaginate [Benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -octyl-D-asparaginate]. -14.1 (c 0.8, CHCl3). 2-3-4. Preparation of Fmoc-D-Asp(OCT)-OH Fmoc-D-Asp(OCT)-OH was prepared using the same method as the Fmoc-L-Asp(OCT)-OH preparation method (Experimental Example 2-2-4). The chemical formula of Fmoc-D-Asp(OCT)-OH is as follows, and the IUPAC name is Benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -Octyl-D-asparagine [Benzyl N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 4 -octyl-D-asparagine]. +7.81 (c 1.5, DMSO). 2-4. Polypeptide synthesis The polypeptide was synthesized as follows. Rink amide AM resin LL (100-200 mesh), a special type of resin with low substitution, was composed of a modified rink amide linker introduced at 0.1 mmol / g into aminomethylpolystyrene (Sigma Aldrich, Nobabichem, 855120). The resin was swollen in 3 mL of DMF for 30 min on a Poly-Prep chromatography column (Bio-Rad) and then subjected to cycles of Fmoc-α-amino acid coupling. A single coupling cycle consisted of two treatment steps, including the removal of the Fmoc protecting group and the subsequent coupling of the amino acid. In each cycle, the Fmoc group was removed with 3 mL of 20% piperidine in DMF under stirring for 15 min, and the resin was washed three times with 3 mL of DMF on a Poly-Prep column. Afterwards, the resin was coupled with a mixture of Fmoc-L-Asp(OtBu) (4 equiv), Fmoc-D-Asp(OtBu) (4 equiv), Fmoc-L-Asp(OCT)-OH (3 equiv), or Fmoc-D-Asp(OCT)-OH (3 equiv) together with HATU (identical to Fmoc-amino acid) and DIEA (6 equiv). The resin containing the mixture in the column was shaken for 30 min for the coupling process, and the resin was washed three times with 3 mL of DMF. After 8, 12, 16, and 20 cycles of binding to the resin, octameric, dodecameric, hexadecameric, and icosameric polypeptides were treated with diethylenetriamine (DET), and the resin was stirred in a 50% DET solution (5 mL) at 4°C for 48 h, then replaced with fresh DET solution four times. The resulting peptide analogs were eluted with a mixture of TFA (90%), DCM (5%), triisopropylsilane (2.5%), and water (2.The peptide analogues were separated from the resin with 2 mL of a TFA cocktail consisting of 5% acetonitrile. The peptide analogues were separated by precipitation in diethyl ether and centrifugation, and their masses were measured by mass spectrometry (Thermo Q Exactive Focus or Waters ZQ 4000). 2-5. High-purity polypeptide analysis The purity of the polypeptide was analyzed by HPLC (KNAUER, Germany, comprised of Smart time manager 5000 with degasser 10ml; LPG (E43OV2, 104107), Smart time pump 1000 including 10ml pump head titanium inlays (EA4300V1, 95270) and UV detector 2500 (E4310, 103886)) using a PFP (pentafluorophenyl) column (Phenomenex, US, PNO 00F-4447-EQ, Luna 3u PFP(2) 100Å, 150x4.60mm, SNO 438270-1). [A = H2O + 0.1% TFA, B = ACN + 0.1% TFA] gradient; 0~5 min (5% B), ~25 min (95% B), flow rate 2 ml / min. 2-6. Polypeptide purification Purification of the polypeptide was performed by HPLC (KNAUR, Germany) using a 3 μm Evosphere PFP semi-prep column (Fortis, UK, size 250x10 mm, SN P01232305-2, PN EVOPFP-100903). [A = H2O + 0.1% TFA, B = ACN + 0.1% TFA] gradient; 0–5 min (5% B), ~25 min (95% B), flow rate 5.5 ml / min. [Experimental Example 3] DDOODDOO Enantiomer Analysis The optical purity of DDOODDOO was analyzed by gas chromatography-mass spectrometry (GC-MS) by HiPep Laboratories (Kyoto, Japan) using an enantiomer labeling method. The polypeptide was hydrolyzed in 6N HCl in D2O, and the residue was acylated using trifluoroacetic anhydride or pentafluoropropionic anhydride. The dissolved residue was injected into GC-MS. The enantiomer purity (% D enantiomer) was calculated as area of ​​D enantiomer / (area of ​​D enantiomer + area of ​​L enantiomer) × 100. [Experimental Example 4] Analysis of the degree of protonation Octameric polypeptide (20 mg) was dissolved in 50 mL of 0.005 M HCl containing 150 mM NaCl and titrated with 0.025 M NaOH containing 150 mM NaCl at room temperature. An automatic titrator (COM-A19, Hiranuma, Kyoto, Japan) was used for the titration. After the pH value in the solution stabilized, the titrant was added in an automated amount of 0.05–0.5 mL. The relationship between pH and the degree of protonation of the polycation (α) was calculated from the obtained titration curve. [Experimental Example 5] Light Scattering Analysis The light-scattered intensity of the polypeptides was measured by static light scattering at 25°C and a detection angle of 173° using a Zetasizer Pro (Malvern Panalytical, Worcestershire, UK) equipped with a He-Ne laser (λ=633 nm). Samples in 10 mM HEPES buffer (pH 7.3) were analyzed using a low-volume quartz cuvette (ZEN2112, Malvern Panalytical). [Experimental Example 6] Circular Dichroism Analysis The secondary structure of the peptide was measured by circular dichroism (CD) spectroscopy (J-815, Jasco, Japan) at room temperature. Polypeptides at a concentration of 0.5 mg / mL in 10 mM sodium phosphate buffer (pH 7.0) were analyzed using a cuvette (path length: 10 mm, Hellma, Mullheim, Germany). [Experimental Example 7] Manufacturing and Characterization of Polyplexes Loaded with FLuc-mRNA Octameric polypeptide was first dissolved in 0.01 M HCl at a concentration of 4 mg / mL and the counterion was exchanged from trifluoroacetate to chloride by stirring at 4°C for 1 h. 10 mM HEPES buffer (pH 7.3) was then added to the polypeptide solution to obtain a concentration of 2 mg / mL. The octameric polypeptide was further diluted in 10 mM HEPES buffer (pH 7.3) and then mixed with the FLuc-mRNA solution (100 ng / μL mRNA in 10 mM HEPES buffer, pH 7.3) to produce mRNA-loaded polyplexes (20 ng / μL mRNA) with the desired molar ratio of amino groups of the DET moiety to phosphate groups of the FLuc-mRNA at the desired N / P ratio. DDOO polypeptides of 12-mer, 16-mer, and 20-mer lengths were dialyzed against 0.01 M HCl to exchange the counterion from acetate to chloride. Finally, the polypeptides were dialyzed against deionized water and lyophilized. For further experiments, the polypeptides were dissolved in 10 mM HEPES buffer (pH 7.3). Polyplex size (cumulant diameter) and size distribution (polydispersity index (PDI)) were measured by dynamic light scattering using a Zetasizer Pro (Red) equipped with a He-Ne laser (λ=633 nm) at 25°C and a detection angle of 173°. A low-volume quartz cuvette (ZEN2112) was used for size measurements. Zeta-potential of polyplexes was measured using the same device at 25°C using a folded capillary cell (DTS1070, Malvern Panalytical). Zeta-potential was calculated from the measured electrophoretic mobility based on the Smoluchowski equation. Optical microscopy (Axio Observer, Carl Zeiss, Germany) was used to analyze the macroscopic properties of polyplexes. DDOO of 8-mer or 20-mer in 10 mM HEPES buffer (pH 7.3) was mixed with 20-nucleotide-long ssRNA (100 ng / μL ssRNA in 10 mM HEPES buffer, pH 7.3) at N / P=5 for 1 h. Polyplexes (10 μL, 20 ng / μL ssRNA) were imaged through 8-well Lab-Tek chambered borosilicate coverslips (Nalge Nunc International, Rochester, NY) at room temperature. [Experimental Example 8] Gel Retardation Analysis Polypeptides and FLuc-mRNA were mixed at the desired N / P ratio as described above. The polyplex solution was mixed with glycerol (final glycerol concentration: 8 vol%, final mRNA amount: 100 ng), and the samples were electrophoresed on an agarose gel (1.2 wt% agarose gel, 1× TBE buffer, 135 V, 15 min). The mRNA in the gel was stained with ethidium bromide and visualized using a WSE-5300 Printgraph CMOS (ATTO, Tokyo, Japan). To determine the stability of polyplexes in PBS containing 10% FBS (PBS / FBS), polyplexes (N / P=5, 100 ng, 10 μL) were mixed with PBS / FBS (90 μL) at 37°C for 1 h, purified using the RNeasy Mini Kit (Qiagen), and the samples were subjected to electrophoresis on an agarose gel (1.2 wt% agarose gel, 1× TBE buffer, 135 V, 15 min). The mRNA in the gel was then stained with ethidium bromide and visualized using a WSE-5300 Printgraph CMOS. [Experimental Example 9] Polyplex mRNA Transfection A549 cells were seeded in 96-well plates at a density of 8,000 cells / well in DMEM medium containing 10% FBS (DMEM / FBS). The next day, polyplex solutions prepared from FLuc-mRNA were added to each well at various N / P ratios or mRNA concentrations, and the cells were cultured for 24 h. The expression level of firefly luciferase was measured by photoluminescence intensity of cell lysates using the Luciferase Assay System (Promega, Madison, WI), and the photoluminescence intensity was measured with a luminescence microplate reader (Mithras LB 963 Centro, Bertold Technologies GmbH & Co.KG, Bad Wildbad, Germany). [Experimental Example 10] Flow cytometry A549 cells were seeded in 24-well plates at a density of 50,000 cells / well in DMEM / FBS, and the medium was replaced with fresh DMEM / FBS the following day. Polyplex solutions (N / P=5) containing Cy5-labeled FLuc-mRNA (Cy5-mRNA) were then added to each well (500 ng mRNA / well). After 4 h of incubation, the medium was removed, and the cells were washed twice with 1 mL of PBS. The cells were treated with trypsin-EDTA solution for 1 min and suspended in PBS. The fluorescence intensity of the cell-derived Cy5-mRNA was measured using a CytoFLEX (Beckman Coolter, CA, USA). Cells were excited with a 638 nm laser. The emitted fluorescence was detected using an APC channel with a 660 / 10 BP filter. [Experimental Example 11] Isothermal calorimetry measurement Isothermal titration calorimeter (ITC) measurements were performed as follows. 20-nucleotide ssRNA (39.6 μM) and DDOO polypeptide (396 μM) were dissolved in 10 mM HEPES buffer (pH 7.3). The exotherm during polyplex complexation between the two samples was measured using a MicroCal Auto-iTC200 (Malvern Panalytical, USA). The polypeptide sample was injected into the RNA solution for 4 s at 150-s intervals at 25°C. A total of 19 drops were injected to calculate the enthalpy and entropy for complexation between RNA and peptide. Data were analyzed using MicroCal Origin 7.0 software. [Experimental Example 12] MD Simulation Cationic polypeptides were parameterized using the Antechamber package in AmberTools19 for atomistic MD simulations (AAMD), using partial charges assigned using the Gasteiger method. AAMD simulations were performed with Amber ff14SB in GROMACS 2021.4. To confirm the polypeptide structure, the cationic polypeptide was first immersed in a simple point charge (SPC) water chamber, to which chloride counterions were added to neutralize the system's charge. The system was energy minimized and equilibrated for 100 ps in the NVT ensemble at 300 K using a V-scale thermostat. This was followed by a Parrinello-Rahman barostat.

[0029] The NPT equilibrium was achieved at 1.0 bar for 100 ps using a Parrinello Rahman barostat. Afterwards, the NPT equilibrium was achieved at 1.0 bar for 100 ps. Finally, the system was simulated for 100 ns without constraints. For the complex simulation, the RNA (sequence: AUGGA GGACG CCAAG AACAU) and the cationic polypeptide were placed in an SPC water box 2 nm away from the RNA, and the system was neutralized by adding sodium counterions. The system was then minimized, equilibrated as mentioned above, and simulated for 100 ns without constraints. [Experimental Example 13] In vitro gene editing efficacy analysis using a reporter system via mRNA delivery. HEK293-loxP-GFP-RFP (Neo) cells were seeded at a density of 8,000 cells / well in DMEM / FBS in 96-well optical bottom plates (165305, ThermoFisher Scientific). HEK293-loxP-GFP-RFP cells contain the LoxP-GFP-stop-LoxP-RFP cassette under the CMV promoter, which fluoresces red after LoxPs are cleaved by Cre recombinase. The medium was replaced with fresh DMEM / FBS the next day. Subsequently, polyplex solution containing Cre-mRNA (N / P = 5) was added to each well (100 ng mRNA / well). After 48 h of incubation, cells were observed using CLSM (ZEISS LSM 980, Carl Zeiss, Oberkochen, Germany) equipped with a 20x objective (Carl Zeiss). The number of GFP or RFP-positive cells and the fluorescence intensity in the images were analyzed using Image J. [Example 1] Sequence-controlled polypeptide synthesis A series of sequence-controlled polypeptides containing DET and OCT moieties were prepared via solid-phase peptide synthesis using tert-butyl and Fmoc-protected aspartic acid (hereinafter referred to as Fmoc-L-Asp(OtBu) or Fmoc-D-Asp(OtBu)) and octyl-modified aspartic acid (hereinafter referred to as Fmoc-L-Asp(OCT)-OH or Fmoc-D-Asp(OCT)-OH) (Fig. 1). For the synthesis of Fmoc-L-Asp(OtBu), the carboxyl group of Fmoc-L-Asp(OtBu)-OBn was protected via a benzyl ester reaction to generate Fmoc-L-Asp(OtBu)-OBn. The tert-butyl group was deprotected by treatment with trifluoroacetic acid, and 1-octylamine was conjugated to Fmoc-L-Asp(OH)-OBn using HATU reagent. Subsequently, Fmoc-L-Asp(OCT)-OH was generated through deprotection of the benzyl ester using hydrogen (H2) in a Pd / C catalyst. To confirm that Fmoc-L-Asp(OH)-OBn and Fmoc-L-Asp(OCT)-OH were successfully synthesized, 1 H NMR and 13C NMR, IR spectroscopy, and mass spectrometry were performed (Figs. 2 and 3). Fmoc-D-Asp(OCT)-OH was also synthesized using a similar protocol using Fmoc-D-Asp(OtBu). Various polypeptides were synthesized on resin, and precursor polypeptides were generated through repeated coupling cycles of Fmoc-L / D-Asp(OtBu) or Fmoc-L / D-Asp(OCT)-OH. The precursor polypeptides were then reacted with an excess of DET to convert Asp(OtBu) to Asp(DET). L-Asp(DET) and L-Asp(OCT) units were denoted as D and O, respectively, and D-Asp(DET) and D-Asp(OCT) units were denoted as (D) and (O), respectively. Two alternative copolypeptides (DODODO and DDOODDOO), one diblock copolypeptide (DDDDOOOO), and one homopolypeptide (DDDDDDD) were synthesized as octameric polypeptides. Three alternative copolypeptides (DD00) with longer lengths (12mer, 16mer, and 20mer) than the alternative copolypeptide (DDOODDOO) were also prepared to analyze the effect of polypeptide length on mRNA complexation and in vitro transfection. The structures and abbreviations of each polypeptide are summarized in Fig. 7. Subsequently, the D / L-20mer DDOO was synthesized to produce 20mer DDO(O), (D)DOO, (D)(D)OO, and (D)D(0)O. Successful synthesis of the sequence-controlled polypeptides was confirmed by mass spectrometry (Figs. 4 and 6). The purity of the polypeptide was analyzed using HPLC in a gradient solvent, confirming a single peak of the product (Figures 5 and 6). The optical purity of the octameric DDOODDOO was confirmed by GC-MS analysis preprocessed with an enantiomer labeling method. As a result, DDOODDOO had a purity of 98.Containing 6% L-form Asp, it was confirmed that DET treatment of the precursor polypeptide on the resin did not interfere with the optical purity (Fig. 8). [Example 2] Analysis of the degree of protonation and critical binding concentration of polypeptides 2-1. Protonation degree analysis First, the degree of protonation of the amino group in the side chain of the octameric polypeptides was analyzed using automated potentiometric titration. Three octameric copolypeptides and one homopolypeptide, used as controls, were dissolved in acidic buffer (pH 2) in the presence of 150 mM NaCl, and the pH of the solution was measured by adding NaOH (Figure 9A). As a result, the three copolypeptides showed different protonation degrees. DODODODO and DDDDOOOO showed lower buffering capacities from pH 5 to pH 7 compared to DDOODDOO, confirming that the sequence arrangement of the amino group significantly affects the degree of protonation. In the case of DODODODO, it was confirmed that the hydrophobic octyl moiety adjacent to the amino group can induce a low buffering capacity by preventing the influx / efflux of proton ions. Diblock copolypeptide (DDDDOOOO) readily aggregated in aqueous solution, inhibiting the protonation of its amino groups. In contrast, DDOODDOO exhibited a degree of protonation similar to that of the homopolypeptide (DDDDDDDD), which has been widely studied for its high buffering capacity and endosomal escape ability after cellular internalization. This suggests that at least two amino moieties must be separated from the hydrophobic moiety to facilitate proton influx / efflux toward the polypeptide. 2-2. Critical binding concentration analysis The critical association concentration (CAC) of peptides measured by static light scattering was analyzed. Polypeptides at various concentrations (0.001–4 mg / mL) were dissolved in 10 mM HEPES buffer (pH 7.3), and the CAC of each polypeptide was calculated after measuring the scattered light intensity of each sample. As a result, DDOODDOO (CAC = 0.304 mg / mL) showed a CAC that was approximately 2–3 times higher than that of DODODODO (CAC = 0.155 mg / mL) and DDDDOOOO (CAC = 0.083 mg / mL) (Fig. 9B). The homopolypeptide (DDDDDDDD), used as a control, was well dissolved in the buffer (pH 7.3), but no CAC was observed up to a treatment concentration of 4 mg / mL. The low CAC of DODODODO and DDDDOOOO confirmed that the degree of protonation of these polypeptides was not efficient. On the other hand, alternative polypeptides with longer lengths (12-mer, 16-mer, and 20-mer DDO) showed CAC similar to that of the octameric DDOO, confirming that the length of the polypeptide did not affect the CAC (Figure 9C). From the above results, it is clear that the sequence arrangement of the polypeptides affects the influx / efflux of proton ions or the intermolecular interactions of each polypeptide in the buffer, thereby affecting the pK a It was confirmed that it had a significant effect on the value and CAC (or water solubility). [Example 3] Polypeptide conformation analysis The conformation of the octameric polypeptide was analyzed using circular dichroism (CD) spectroscopy at a concentration of 0.5 mg / mL (pH 7.0). As a result, the spectra of the octameric polypeptides appeared as a single broad curve with an upper peak at 205–210 nm, regardless of the sequence arrangement (Figure 9D). The spectra were analyzed using CDSSTR software, and all octameric polypeptides used adopted a β-turn (a β-turn is a non-canonical peptide secondary structure containing four consecutive amino acid residues characterized by an intramolecular hydrogen bond between the first and third residues and a 7 Å distance between α-carbons). To determine how these polypeptides adopt this non-canonical secondary structure, we performed all-atom molecular dynamics (AAMD) simulations. After equilibrating the polypeptides in a salt-neutralized water bath for 50 ns, we confirmed that all four polypeptides adopted specific β-turn characteristics (Figures 9E and 10). Specifically, the structurally flexible DET and OCT side chains enabled β-turn formation without excessive deformation of the polypeptide backbone. Furthermore, the DET side chains were clearly observed to be well exposed to the outside in all three copolypeptides, confirming that protons could freely enter and exit without being restricted by their own secondary structure. Instead, in DDDDOOOO and DODODODO, the four OCT side chains were aligned in the same direction, inducing intermolecular hydrophobic interactions. This supports that the above polypeptides (DDDDOOOO and DODODODO) exhibit lower CAC values ​​than DDOODDOO. From the above results, it can be concluded that intermolecular bonds can inhibit the smooth inflow / outflow of proton ions, and the pK of the polypeptides aIt was confirmed that the intermolecular bonds between polypeptides are determined by the arrangement of hydrophobic side chains. [Example 4] Analysis of physicochemical properties and mRNA delivery efficiency of polyplexes. 4-1. Analysis of free mRNA bands according to N / P ratio by polyplex Polyplex samples were prepared by mixing FLuc-mRNAs with 1929 nucleotides at various N / P ratios in 10 mM HEPES buffer (pH 7.3), and their complexes were confirmed by agarose gel electrophoresis (Fig. 11). Among the four octameric polypeptides, the free mRNA band (mRNA not bound to the polymer) disappeared in DDDDDDDDD at N / P≥2, which is thought to be because the DET moiety in the side chain has a degree of amine protonation of approximately 50%, and an N / P of 2 corresponds to the charge neutralization point between the protonated amine of the DET moiety and the phosphate of FLUC-mRNA. In addition, DDOODDOO showed a similar association of mRNA with the homopolypeptide, in which the free mRNA band disappeared at N / P≥2. This is in good agreement with the degree of protonation of DDOODDOO shown in Figure 9A, indicating that the two repeating units of DET are sufficiently associated with their counterparts. On the other hand, the free mRNA band disappeared in DODODODO and DDDDOOOO at N / P≥15 and N / P≥7.5, respectively, which is thought to be due to the small amount of accessible protonated amino groups in the polypeptide. DODODODO, which has one repeating unit of DET, had the lowest binding affinity for mRNA, indicating the importance of alternative sequence arrangements for mRNA binding. DDOO, which has a long length, showed the disappearance of the free mRNA band at N / P≥2, confirming that the degree of protonation of the polypeptide and its association with mRNA are not affected by the length of the polypeptide. 4-2. Analysis of hydrodynamic diameter, polydispersity index, and zeta potential according to N / P ratio of polyplex Hydrodynamic diameter (D) of FLUC-mRNA loaded polyplex samples prepared at various N / P ratios H The polydispersity index (PDI) and zeta potential were measured using a Zetasizer (Table 1). The delayed mRNA association of DODODODO and DDDDOOOO was also confirmed through zeta potential. As a result, at N / P=5, the zeta potential of DODODODO was -2.6±2.2 mV and DDDDOOOO was -16±4.9 mV, whereas DODODODO was +16±0 mV. In addition, as the length of the DDOO polypeptide increased, the size of the polyplex decreased. The hydrodynamic diameter of the 20-mer DDOO polyplex was ~200 nm. The zeta potentials of all polyplexes with 12-mer, 16-mer, and 20-mer DDOO polypeptides were between +30 and +40 mV. polypeptidesN / P ratioD H(nm)PDIzeta-potential (mV)DODODODO5450 ± 3600.55 ± 0.17-2.6 ± 2.210450 ± 2000.57 ± 0.08-3.0 ± 9.0203200 ± 6600.55 ± 0.17-1.1 ± 3.3304330 ± 4300.35 ± 0.130.1 ± 6.6DDOODDOO55720 ± 20100.45 ± 0.0516 ± 010950 ± 5700.88 ± 0.0938 ± 4.720400 ± 300.86 ± 0.0438 ± 4.430460 ± 2000.51 ± 0.0739 ± 4.1DDDDOOOO5370 ± 1100.54 ± 0.11-16 ± 4.910400 ± 400.59 ± 0.079.0 ± 6.3202660 ± 9500.60 ± 0.1527 ± 3.9304280 ± 6800.41 ± 0.0638 ± 3.812mer DDOO5430 ± 2100.46 ± 0.1531 ± 4.316mer DDOO5130 ± 70.34 ± 0.0636 ± 3.820mer DDOO5200 ± 200.36 ± 0.1233 ± 4.320mer DDO(0)56900 ± 18000.40 ± 0.1326 ± 3.420mer (D)DOO5190 ± 200.55 ± 0.0831 ± 4.020mer (D)(D)OO5200 ± 300.64 ± 0.1531 ± 4.920mer (D)D(0)O5170 ± 250.43 ± 0.0633 ± 5.1 * Table 1 above is expressed as mean±SD (n=5). * In the above experimental group, D or O in parentheses is D-form, and D or O not in parentheses is L-form. Parentheses are used only to indicate D-form. 4-3. Analysis of mRNA delivery efficiency (mRNA translation efficiency) The intravenous (IVT) mRNA delivery efficiency of the polyplex was evaluated using A549 cells. Polyplex samples were generated by mixing the octameric polypeptide with FLuc-mRNA at various N / P ratios. The polyplex samples were transfected into cells at 500 ng mRNA / well. The cells were cultured for 24 h, and FLuc expression levels were measured using a luminescence plate reader (Figure 12A). As a result, at N / P=30, the DDOODDOO polyplex exhibited significantly higher luciferase expression levels than the other polypeptide polyplexes. This confirms that DDOODDOO, which has an appropriate pKa for endosomal escape and a hydrophobic moiety for high polyplex stability, is essential for high mRNA translation efficiency. In addition, experiments were also performed on longer DDOO polypeptides. Polyplex samples were generated by mixing 12-mer, 16-mer, and 20-mer DDOO polypeptides with FLuc-mRNA at N / P=5, and the polyplex samples were transfected into A549 cells at various mRNA concentrations (Fig. 12B). As a result, the luminescence intensity of the longer DDOO polyplexes was 10 times higher than that of the octameric DDOO polyplexes at N / P=30 (500 ng mRNA / well). 3 ~10 4 This revealed that the polypeptide length affects the mRNA transfection efficiency. Furthermore, to elucidate the underlying mechanism of polypeptide length-dependent IVT mRNA expression, we analyzed the cellular uptake of IVT mRNA by flow cytometry using Cy5-labeled FLuc-mRNA (Cy5-mRNA). Prior to performing flow cytometry, A549 cells were incubated with naked Cy5-mRNA or Cy5-mRNA-loaded polyplexes for 4 h ( Figures 12C and 13 ). As a result, polyplexes using 12-, 16-, and 20-mer DDOO showed 5- to 7-fold higher uptake than polyplexes using DDOODDOO, confirming that polypeptide length affects cellular uptake. Additionally, the stability of the polyplex (or IVT mRNA payload) was analyzed after incubation with 10% FBS at 37°C for 1 hour (Fig. 12D). The polyplexes were then dissociated, and intact IVT mRNA was visualized by agarose gel electrophoresis. Results showed that a greater amount of intact IVT mRNA was present in the longer DDOO polypeptide. This confirmed that the efficient cellular uptake of the polyplexes was primarily due to their high tolerability in the FBS solution. In addition, to determine the effect of isomers (L-form, D-form) on mRNA delivery efficiency, A549 cells were transfected with polyplexes containing various FLuc-mRNAs (Fig. 12E). As a result, 20-mer (D)DOO showed a luminescence intensity 2.4 times higher than that of 20-mer DDOO, which is the L-form. From the results, it was confirmed that the isomers of the polypeptide affect the mRNA delivery efficiency. In addition, 20-mer DDO(O) and (D)D(O)O showed 10 times higher luminescence intensity than 20-mer (D)DOO. 1 ~10 2 It was confirmed that insertion of D-Asp(OCT) significantly reduced mRNA translation efficiency, as shown by the low luminescence intensity. In addition, the in vitro gene editing efficacy was analyzed using HEK293-loxP-GFP-RFP (Neo) cells, which exhibit red fluorescence after LoxPs are cleaved by Cre recombinase (Fig. 14A). 20-mer DDDO and (D)DDO were mixed with Cre-mRNA to form polyplex samples at N / P=5. Lipofectamine 3000 was used as a positive control. The samples were transfected into cells for 48 h, and the cells were observed using CLSM. The amount and fluorescence intensity of GFP and / or RFP-positive cells were quantified among 100 randomly selected cells. As a result, cells that were not treated with the samples expressed a strong green fluorescence signal, and downstream RFP was not expressed (Fig. 15A). The 20-mer DDDO exhibited higher gene editing efficacy than the 20-mer (D)DOO and the positive control. Specifically, cells treated with 20-mer DDDO polyplex showed 82 RFP-positive cells with reduced GPF fluorescence intensity (Figs. 14B and 14C). Cells treated with 20-mer (D)DOO polyplex or positive control showed 43 and 55 RFP-positive cells, respectively (Figs. 15B and 15C). From the above results, it was confirmed that 20-mer DDOO showed higher delivery efficiency of Cre-mRNA than commercially available transfection reagents, and 20-mer DDDO showed higher delivery efficiency of Cre-mRNA than 20-mer (D)DOO. This is thought to be due to the different mRNA lengths of FLuc- and Cre-mRNA (1929 and 1351 nt, respectively), which affect polyplex formation. [Example 5] Length-dependent polypeptide / RNA complexation Because the formation of supramolecular networks within mRNA polyplexes is crucial for improving transfection efficiency, we analyzed the ssRNA complexation process to understand the length-dependent efficacy of DDOO polyplexes. Given that the single-stranded region of mRNA possesses a highly flexible structure and is optimal for polycation bundling, we selected the first 20 nucleotides of Fluc mRNA as a single-stranded RNA (ssRNA) prototype to study the complexation process. To demonstrate the representativeness of this prototype, we analyzed the physicochemical properties (DH, PDI, and zeta potential) of ssRNA-loaded polyplexes similar to those of FLuc-mRNA-loaded polyplexes (Table 2). polypeptidesN / P ratioD H (nm)PDIzeta-potential (mV)DODODODO5260 ± 730.52 ± 0.15-33 ± 410570 ± 4100.62 ± 0.22-19 ± 19201660 ± 2470.47 ± 0.12-11 ± 7303180 ± 7090.36 ± 0.140.3 ± 10DDOODDOO51430 ± 3570.45 ± 0.135.8 ± 410700 ± 2410.69 ± 0.1014 ± 420468 ± 910.44 ± 0.0527 ± 330449 ± 1260.41 ± 0.1028 ± 3DDDDOOOO58370 ± 30700.57 ± 0.14-0.2 ± 7105980 ± 14000.42 ± 0.0924 ± 7201680 ± 6260.92 ± 0.0733 ± 1630963 ± 2530.87 ± 0.0731 ± 1112mer DDOO5220 ± 280.24 ± 0.0220 ± 1416mer DDOO5250 ± 310.30 ± 0.0934 ± 1120mer DDOO5260 ± 250.31 ± 0.0433 ± 13 * Table 2 above is expressed as mean±SD (n=5).First, we analyzed the complexation between DDOO polypeptides and ssRNA using isothermal titration calorimetry (ITC). All DDOO polypeptides exhibited exothermic complexation with ssRNA (Figures 16A–16D, Table 3). Furthermore, heat release (ΔH) increased as the DDOO polypeptide length increased from 8 mer to 12 mer, but relatively no significant change was observed when the length increased from 8 mer to 16 mer and 20 mer. This confirmed that DDOO polypeptides can form more energetically stable complexes with ssRNA when the sequence length exceeds 8 mer. In addition to this energy stability, we also confirmed changes in the ssRNA complexation mechanism when the DDOO polypeptide sequence length exceeds 8 mer. Using a single-compartment binding model, a good fit was obtained to the experimental binding isotherm of an 8-mer DDOO / ssRNA pair with a binding stoichiometry (N value) of ~1 ( Figure 16A ). This confirmed that complexation of the 8-mer DDOO / ssRNA is a single-step process. In contrast, the experimental binding isotherms of longer DDOO / ssRNA pairs exhibited two energy transfer steps (red for the first step and green for the second step) ( Figures 16B–D ). This two-step polyelectrolyte complexation process has been previously confirmed in other studies, where the first step corresponds to spontaneous ion pairing of oppositely charged groups, whereas the second step corresponds to the formation of a supramolecular network from the ion pairs. This mechanistic difference is also reflected in the increased N value of the 12-mer compared to the 8-mer. In general, longer DDOO polypeptides have a greater number of cationic groups per molecule and therefore have lower N values ​​when the concentration of the injectant (DDOO polypeptide) is held constant.This trend was also confirmed by the decrease in N values ​​when the DDOO polypeptide was extended from 12 mer to 20 mer. However, the opposite pattern was observed when the length increased from 8 mer to 12 mer, with N values ​​increasing rather than decreasing. This higher-than-expected N value is thought to be due to the consumption of 12 mers in forming a supramolecular network that demonstrates the mechanistic transition from the first step to the second step ssRNA complex when the DDOO polypeptide exceeds 8 mer length. polyplexesNK (M-1)ΔH (kcal / mol)TΔS (kcal / mol)8mer DDOO / 20nt ssRNA0.93 ± 0.011.74×10 6 ±2.91×10 5 -32.4 ± 0.5-23.912mer DDOO / 20nt ssRNA1.53 ± 0.025.14×10 6 ±1.66×10 6 -59.4 ± 1.0-50.416mer DDOO / 20nt ssRNA1.07 ± 0.014.40×10 6 ±1.40×10 6 -46.7 ± 1.0-37.520mer DDOO / 20nt ssRNA0.63 ± 0.017.31×10 6 ±2.55×10 6 -66.1 ± 1.4-56.6 Subsequently, AAMD was performed to elucidate the molecular basis for the length-dependent transition of the ssRNA complexation mechanism observed in the ITC measurements. To complement the experimental conditions, the same ssRNA sequence was used in the MD simulations. MD simulations (100 ns) of 2 × 8mer / ssRNA and 1 × 16mer / ssRNA were sampled to compare the ssRNA complexation process between cationic polymers with the same sequence format ((DDOO)n) and residue number (8 × OCT and 8 × DET), and the existence of two different molecular conformations (2 × 8mer vs. 1 × 16mer) was confirmed ( Figure 16E ). The conformation of the DDOO polypeptide was not significantly affected after complexation with ssRNA, which is likely due to the intramolecular hydrogen bond stabilization imposed by multiple β-turns, which are similar to the polypeptide structure ( Figure 9E ). As a result of the unperturbed conformation of the polypeptide, we confirmed that the cationic moiety, DET, was displayed in a defined pattern for ssRNA complexation (Figure 16E). From these AAMD results, it can be assumed that the 8-mer DDOO, which has a compact molecular size due to its secondary structure, is more likely to induce ssRNA complexation (Figure 16E). In contrast, the longer DDO polypeptide with a more extended molecular size could potentially act as a bridge around ssRNA, facilitating the formation of a supramolecular network within the polyplex. Overall, AAMD revealed that the conformation of the DDOO polypeptide is stable when bound to ssRNA, revealing the molecular basis of the length-dependent ssRNA complexation mode. Finally, we analyzed how changes in the complexation mechanism between the DDOO polypeptide and RNA affect the macroscopic properties of the complexes. The DDOO / RNA complexes at NP = 5, which were used in the in vitro mRNA transfection assay, were imaged using bright-field microscopy. As a result, micron-sized aggregates were observed only in the 8mer / ssRNA complexes, while such aggregates were not observed in the ssRNA complexes formed with longer DDOO polypeptides. These results indicate that DDOO polypeptides tend to induce the formation of micron-sized aggregates when complexed with ssRNA through a single ion-pairing step. This result can be explained by the formation of micron-sized aggregates as these charge-neutralizing complexes undergo hydrophobic collapse in the absence of a supramolecular network (Figure 16F). In contrast, longer DDOO / ssRNA complexes with a supramolecular network can form more condensed structures (Figure 16F). While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A polypeptide polymer represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1 n is one of 1 to 20, a and b may be the same or different and are one of 1 to 5, c and d may be the same or different and are one of 1 to 20, R 1 and R 2 may be the same or different, and are hydrogen or (C4-C12)cycloalkyl.

2. In the first paragraph, in the chemical formula 1, n is one of 1 to 5, a and b may be the same or different and are one of 1 to 3, c and d may be the same or different and are one of 5 to 10, and R 1 and R 2 A polypeptide polymer, wherein each may be the same or different and is characterized by being hydrogen or (C4-C8)cycloalkyl.

3. In the first paragraph, in the chemical formula 1, n is one of 1 to 5, a and b are one of 1 to 3, c and d are one of 5 to 10, and R 1 and R 2 A polypeptide polymer characterized by being hydrogen or (C4-C8)cycloalkyl.

4. In the third paragraph, the polymer is a polypeptide polymer characterized in that diethylenetriamine (DET) or octylamine (OCT) is bound to aspartic acid.

5. A polypeptide polymer according to claim 4, characterized in that the form in which diethylenetriamine (DET) or octylamine (OCT) is bound to the aspartic acid is in the L-form or D-form form.

6. A polypeptide polymer according to claim 1, characterized in that the polypeptide polymer increases RNA transcription efficiency.

7. A polypeptide polymer according to claim 6, characterized in that the RNA is at least one selected from the group consisting of mRNA, siRNA, ASO (Antisense oligonucleotide), and gRNA (guide RNA).

8. A polypeptide polymer according to claim 7, characterized in that the mRNA is an in vitro transcribed (IVT) mRNA.

9. A composition for RNA delivery comprising the polypeptide polymer of paragraph 1.

10. A reagent composition for promoting RNA transcription comprising the polypeptide polymer of paragraph 1.

11. A step for producing a conjugate by combining diethylenetriamine (DET) with aspartic acid (step 1); A step (second step) of producing a conjugate in which octylamine (OCT) is combined with aspartic acid; and A method for producing a polypeptide polymer according to claim 1, comprising a step (third step) of combining the conjugate of the first step and the conjugate of the second step.

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