Modified polypeptide based on ARC protein and application of modified polypeptide as nucleic acid delivery carrier

Abstract

The invention provides a polypeptide variant based on ARC protein and application of the polypeptide variant as a nucleic acid delivery carrier, the polypeptide variant based on ARC protein is a variant based on spatial conformation, amino acid point mutation and side chain modification of a lipid group or a lipid group derivative, the spatial conformation is alpha helix, and the amino acid point mutation is alpha helix. The target of the point mutation is to increase the isoelectric point to 10.0 or above. The polypeptide variant has an excellent affinity constant of nucleic acid, can wrap mRNA or DNA and form a structure similar to a virus capsid, not only can protect mRNA from being degraded by nuclease, but also is helpful for releasing mRNA or DNA under the action of lysosome / endosome after a mediating compound enters cells, and can activate and generate effective body fluid and cellular immune response in vivo, so that the polypeptide variant has the advantages of being capable of effectively improving the immunogenicity of the cells and improving the immunogenicity of the cells. The polypeptide has obvious immune protection on rat pox challenge and melanoma, and has good biocompatibility, so that the polypeptide can be prepared into a novel nucleic acid delivery carrier based on bionic polypeptide.

Description

Technical Field

[0001] The present invention relates to a novel nucleic acid delivery vector and its application in mRNA vaccines and gene therapy, belonging to the field of biomedical technology. Background Art

[0002] mRNA vaccines have become an important direction for the development of novel vaccines due to their characteristics such as rapid synthesis and strong immune activation. Since mRNA molecules are easily degraded and difficult to cross the membrane, the vector delivery system is the key core technology of mRNA vaccines. Currently, lipid nanoparticles (LNPs) are the most widely used mRNA vaccine delivery system, mainly composed of 4 lipids, including ionizable lipids or cationic lipids, cholesterol, helper phospholipids, and PEGylated lipids. By using microfluidic technology, the 4 lipids dissolved in the ethanol phase are mixed with the mRNA molecules in the aqueous phase in proportion to achieve the encapsulation of mRNA and form uniform nanoparticles. mRNA vaccines based on LNPs can activate a strong immune response. However, compared with vaccines developed by other technology platforms, clinical data show that the marketed COVID-19 mRNA vaccines have a higher adverse reaction rate, including fever rate, myocarditis, autoimmune diseases, etc. Currently, the LNP is mainly optimized by adjusting the LNP formulation and developing novel ionizable lipids, etc., but the inherent bottleneck of the LNP system has not been fundamentally solved.

[0003] Developing a novel mRNA delivery vector with new concepts, safety, and high efficiency has become an important direction for the development of mRNA innovative technologies. Based on the self-assembly between proteins and nucleic acids, the protein coat of a virus can encapsulate nucleic acids and form natural nanoparticles with high efficient cell infection ability. In addition, mammalian endogenous retrovirus-like proteins such as PEG10, ARC, etc., can also encapsulate their own mRNA and play specific functions in the body through extracellular vesicles. There have been reports on the preparation of virus-like particles that can deliver mRNA by transfecting cells with lentiviral plasmids based on similar virus vector or pseudovirus packaging methods. However, these strategies are inseparable from complex and cumbersome processes such as cell culture and purification, which pose great challenges to quality control.

[0004] The object of the present invention is based on the virus biomimetic concept of protein capsid encapsulating nucleic acids, adopting a technical route different from the LNP that relies on multi-component polymer lipids and the virus-like packaging that relies on cell transfection, and developing a novel single-component vector based on peptides in a controllable self-assembly manner in vitro to achieve efficient encapsulation and delivery of mRNA and apply it to the research and development of novel mRNA vaccines and drugs. Summary of the Invention

[0005] For the above purposes, the present invention first provides a polypeptide variant based on the ARC protein. The polypeptide variant based on the ARC protein is a variant designed based on the spatial conformation, with positive charge modification by amino acid point mutation and side chain modification. The spatial conformation is an α-helix. The target of the positive charge modification by amino acid point mutation is to increase the isoelectric point to above 10.0. The polypeptide variant based on the ARC protein also has side chain modification with a lipid group or a lipid derivative.

[0006] In a preferred embodiment, the amino acid sequence of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.6, 7 or 14. The side chain modification with a lipid group or a lipid derivative in the present invention means that any amino acid residue in the polypeptide variant based on the ARC protein is substituted with a lipid group or a lipid derivative. The lipid group refers to a group composed of an aliphatic hydrocarbon chain (straight or branched chain), usually in the form of -R (such as methyl -CH3, ethyl -C2H5, or a long chain such as hexadecyl -C 16 H 33 ). The lipid derivative refers to a metabolite based on the lipid group, such as triglyceride, cholesterol, phospholipid, glycolipid, etc.

[0007] In a preferred embodiment of the present invention, the lipid group is octadecyl (C18), or the lipid derivative is cholesterol (CHO).

[0008] In a preferred embodiment, the position where the side chain modification with a lipid group or a lipid derivative occurs is at the N-terminus, C-terminus or middle position of the polypeptide variant based on the ARC protein. When the position where the side chain modification with a lipid group or a lipid derivative occurs is at the C-terminus of the polypeptide variant based on the ARC protein, lysine for providing the position of the side chain modification with a lipid group or a lipid derivative is also provided at the C-terminus.

[0009] In a specific embodiment of the present invention, the sequence of the side chain modification with a lipid group of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.14. The lipid group is octadecyl, and the modification position is the C-terminus. In the present invention, the polypeptide variant based on the ARC protein is named "H3M1-4".

[0010] In a preferred embodiment, a linking element is provided between the polypeptide variant based on the ARC protein and the lipid group.

[0011] In a more preferred embodiment, the linking element is GSG and / or polyethylene glycol.

[0012] In a specific embodiment of the present invention, the sequence of the side-chain modification of the lipid group of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.7. The lipid group is octadecyl, the modification position is the C-terminus, and the linking element is GSG-K-PEG8. In the present invention, the polypeptide variant based on the ARC protein is named "H1M6-7".

[0013] In a preferred embodiment of the present invention, the sequence of the side-chain modification of the lipid group of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.14. The lipid group is octadecyl, the substitution position is the N-terminus or the C-terminus, and the linking element is GSG.

[0014] In a specific embodiment of the present invention, when the substitution position is the N-terminus, the substituent is C18, and the linking element is GSG, the polypeptide variant based on the ARC protein is named "H3M1-2".

[0015] In a specific embodiment of the present invention, when the substitution position is the C-terminus, the substituent is C18, and the linking element is GSG, the polypeptide variant based on the ARC protein is named "H3M1-5".

[0016] In a preferred embodiment of the present invention, the polypeptide variant based on the ARC protein is further fused with a polypeptide having a targeting function and / or an adjuvant effect.

[0017] In a more preferred embodiment, the sequence of the polypeptide having a targeting function and / or an adjuvant effect is as shown in SEQ ID NO.22. In the present invention, the polypeptide having the sequence as shown in SEQ ID NO.22 is named 22A.

[0018] In a specific embodiment of the present invention, the polypeptide 22A is fused to the N-terminus of the polypeptide variant based on the ARC protein "H3M1-5", and the fused polypeptide is named "22A-H3M1-5 fusion peptide".

[0019] Secondly, the present invention provides the use of the above-mentioned polypeptide variant based on the ARC protein as a nucleic acid delivery vector. The nucleic acid delivery vector described in the present invention refers to a tool or system that can safely and efficiently introduce exogenous nucleic acids (such as DNA, RNA) into target cells, which is a key technology in gene therapy, vaccine development, and molecular biology research. Its core purpose is to overcome the cell membrane penetration barrier and in vivo degradation problem of nucleic acids, and ensure the effective expression or regulation of functional nucleic acids in target cells.

[0020] In a preferred embodiment of the present invention, the nucleic acid is plasmid DNA or mRNA.

[0021] In a specific embodiment of the present invention, the application is to prepare a gene editing vector.

[0022] In a specific embodiment of the present invention, the application is to prepare an mRNA vaccine.

[0023] The present invention provides a polypeptide variant based on the ARC protein. The variant is derived from a polypeptide with an α-helical conformation in the ARC protein. After introducing point mutations of amino acids for positive charge modification and side chain modification of a lipid or lipid derivative, the affinity constant ( KD ) is greatly improved, which can encapsulate mRNA or DNA and form a structure similar to a viral capsid. It can not only protect mRNA from degradation by nucleases, but also help mediate the release of mRNA under the action of lysosomes / endosomes after the complex enters the cell and initiate the expression of target genes. Moreover, it also has good biocompatibility. Therefore, it can be prepared into a novel nucleic acid delivery vector based on biomimetic polypeptides, which can achieve efficient encapsulation and delivery of in vitro transcribed mRNA and plasmid DNA through self-assembly. The single-component biomimetic polypeptide delivery vector provided by the present invention can simply form a nanostructure with stable structure and uniform particle size with nucleic acids by mixing, and has high delivery efficiency at the cellular and in vivo levels. Delivering mRNA vaccines encoding model antigens such as monkeypox virus M1R and OVA in a mouse model can activate effective humoral and cellular immune responses, and have significant immune protection against mousepox challenge and melanoma. Compared with the four-component liposome nanoparticles (LNP) with strong inflammatory activity and liver enrichment, the mRNA vaccine or gene editing vector delivered by this single-component biomimetic polypeptide has the expression of its target gene limited only to the injection site, and does not activate the production of obvious inflammatory cytokines. Moreover, the polypeptide vector also has the advantage that the delivery elements can be modularly spliced, and can be fused with other carrier polypeptides to further improve the delivery efficiency. The polypeptide variant based on the ARC protein provided by the present invention is expected to provide a safer, more effective, simple and easy-to-use new strategy for the research and development of novel nucleic acid vaccines and gene therapy drugs. Brief Description of the Drawings

[0024] Figure 1 . Technical roadmap for the design, screening and optimization of the biomimetic vector of the present invention; Figure 2 . Prediction of the spatial conformation of candidate polypeptides by Alphafold3; Figure 3 . Detection of the mRNA cell delivery efficiency of the first-round candidate polypeptide vectors; Figure 4 . Detection of the mRNA cell delivery efficiency of the second-round candidate polypeptide vectors; Figure 5. Detection of encapsulation and cellular delivery of ARC recombinant protein to mRNA; Figure 6 . Detection of encapsulation and cellular delivery of TAT polypeptide to mRNA; Figure 7 . Detection of the molecular weight of H3M1-5 by mass spectrometry; Figure 8 . Detection of the cytotoxicity of H3M1-5; Figure 9 . Detection of the particle size and zeta potential of the self-assembled complex of H3M1-5 and mRNA-Fluc; Figure 10 . Detection of the self-assembled complex of H3M1-5 and mRNA-Fluc by transmission electron microscopy; Figure 11 . Detection of the encapsulation efficiency of the H3M1-5@mRNA-Fluc complex; Figure 12 . Gel retardation assay of the H3M1-5@mRNA-Fluc complex; Figure 13 . Detection of the binding kinetics between the polypeptide carrier and mRNA; Figure 14 . Detection of the cellular delivery efficiency of H3M1-5@mRNA-Fluc at different salt ion concentrations; Figure 15 . Detection of the delivery efficiency of H3M1-5@mRNA-Fluc in different cell lines; Figure 16 . Detection of the cellular delivery efficiency of H3M1-5@mRNA-GFP; Figure 17 . Detection of the cellular delivery efficiency of H3M1-5 to circular mRNA; Figure 18 . Detection of the cellular delivery efficiency of H3M1-5 to plasmid DNA; Figure 19 . Detection of the cellular gene editing efficiency of the CRSPR system delivered by H3M1-5; Figure 20 . Detection of the in vivo delivery efficiency of H3M1-5@mRNA-Fluc at different N / P ratios; Figure 21 . Detection of the in vivo delivery efficiency of H3M1-5@mRNA-Fluc at different time points; Figure 22 . Detection of the antibody response of the H3M1-5@mRNA-M1R monkeypox vaccine; Figure 23 . Detection of the cellular immune response of the H3M1-5@mRNA-M1R monkeypox vaccine; Figure 24 . Detection of antibody response to H3M1-5@ mRNA-OVA tumor vaccine; Figure 25 . Detection of cellular response to H3M1-5@ mRNA-OVA tumor vaccine; Figure 26 . Evaluation of the anti-tumor effect of H3M1-5@ mRNA-OVA in a melanoma model; Figure 27 . Detection of antibody response to 22A-H3M1-5@ mRNA-M1R monkeypox vaccine; Figure 28 . Lethal challenge protection test of 22A-H3M1-5@ mRNA-M1R monkeypox vaccine; Figure 29 . Detection of cytokine levels after immunization with LNP and polypeptide carrier mRNA-OVA vaccines; Figure 30 . In vivo biodistribution detection after LNP and polypeptide carrier delivery of mRNA-Fluc; Figure 31 . Blood biochemical detection after LNP and polypeptide carrier delivery of mRNA-M1R. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, these embodiments are merely exemplary and do not constitute any limitation to the protection scope defined by the claims of the present invention.

[0026] Example 1. Design, screening and optimization of biomimetic carriers Figure 1 The technical roadmap for the design, screening and optimization of the biomimetic carrier of the present invention is given. First, the polypeptide carrier design based on α-helix and isoelectric point is carried out to obtain candidate polypeptide variants based on the ARC protein, and then it is packaged with linearized mRNA encoding luciferase to form a virus-like capsid structure. Then, the first round of screening of candidate polypeptides based on fluorescence intensity is carried out. Next, the candidate polypeptides selected in the first round are lipid-modified, and the second round of screening of candidate polypeptides based on fluorescence intensity is carried out. Finally, polypeptide variants based on the ARC protein are obtained.

[0027] 1. Design and screening of polypeptide variants of ARC protein (1) Polypeptide carrier design based on α-helix and isoelectric point The structures of candidate proteins are analyzed by auxiliary tools such as Alphafold3. Taking the ARC protein as an example, polypeptides with α-helix conformation and isoelectric point greater than 7.0 are selected, namely ARC 99-126(H1), ARC 249-257 (H2), ARC 297-313 (H3) and ARC 329-339 (H4). Based on the H1-H4 polypeptides, the amino acid sequence was modified by combining electrostatic and amphiphilic properties. While maintaining the α-helix conformation, the isoelectric point of the candidate polypeptides was increased to greater than 10.0 to enhance the interaction with mRNA. The design parameters included: j, the number of amino acid point mutations; k, the tandem mode of the repeat sequence (forward or reverse), etc. A total of 18 candidate polypeptides were designed and chemically synthesized (Sangon Biotech) as shown in Table 1. AlphaFold3 prediction indicated that most candidate polypeptides could maintain the α-helix (see Figure 2 ), and the sequence modification did not cause significant changes to the basic conformation of the original polypeptides.

[0028] Table 1. Design of the first-round candidate polypeptides

[0029] (2) Screening of the first-round candidate polypeptide carriers In the cell model, the delivery efficiency of the first-round candidate polypeptides to mRNA was detected. DC2.4 cells (20,000 cells / well, 100 μL) were cultured overnight in a 96-well plate (37 °C, 5% CO2), and the culture medium was DMEM medium containing 10% fetal bovine serum (FBS). The candidate polypeptides were mixed with linearized mRNA encoding luciferase (mRNA-Fluc, purchased from Novoprotein) at an N / P ratio of 20:1 in 20 mM HEPES (pH 7.4) buffer and allowed to interact for 30 min. Before cell transfection, the medium was changed to Opti-MEM serum-free medium, and 125 ng mRNA (n = 3) was added to each well. After 4 hours of cell transfection, the medium was changed back to DMEM + 10% FBS medium. The commercial transfection reagent TransIT ® (Mirus) / mRNA-Fluc mixture and naked mRNA were used as positive and negative controls, respectively. After 24 hours of transfection, the luciferin substrate was added to the cell culture plate to detect the fluorescence intensity. Statistical analysis (one-way ANOVA and Tukey's multiple comparison) showed that the delivery efficiencies of H1M6 (SEQ ID NO.7), H3M1 (SEQ ID NO.14), and H1M5 (SEQ ID NO.6) to mRNA were significantly higher than those of the negative control, while the original polypeptides H1-H4 from ARC did not produce positive results (see Figure 3 ).

[0030] (3) Optimization of the candidate polypeptide carriers based on lipid modification To further enhance the hydrophobicity of the candidate polypeptides and their interaction with cell membranes, thereby improving the cellular delivery efficiency of mRNA, the candidate polypeptides H1M6 and H3M1 selected in the first round were preferably selected and lipid-modified. The optimization parameters included: j, the type of modified lipid such as octadecyl (C18) or cholesterol (CHO); k, the modification position such as the N-terminus, C-terminus or middle position of the candidate polypeptide; l, whether there was a linker such as GSG (glycine-serine-glycine) or polyethylene glycol (PEG) between the candidate polypeptide and the modified lipid group. As shown in Table 2, 18 candidate polypeptides were designed and chemically synthesized (Sangon Biotech).

[0031] Table 2. Design of candidate polypeptides in the second round

[0032] (4) Screening of candidate polypeptide carriers in the second round In a serum-containing cell transfection model, the delivery efficiency of the candidate polypeptides in the second round on mRNA was detected. DC2.4 cells (20,000 cells / well, 100 μL) were cultured overnight in a 96-well plate (37 °C, 5% CO2), and the culture medium was DMEM containing 10% fetal bovine serum. The candidate polypeptide and mRNA-luc were mixed at N / P ratios of 2, 4, and 8 in 20 mM HEPES (pH 7.4) buffer, and after interacting for 30 min, the cells were transfected (n = 3, 125 ng mRNA / well). After 24 hours, a luciferin substrate was added to the cell culture plate to detect the fluorescence intensity. The results (see Figure 4 ) showed that under the condition of 10% serum, H1M6-7, H3M1-2, H3M1-4, and H3M1-5 all had good mRNA delivery efficiency. Among them, H3M1-5 had the highest mRNA delivery efficiency, and the fluorescence intensity could reach 8×10 7 RLU or more. When the N / P ratio was 2 and 4, the fluorescence intensity was significantly higher than that of the positive control commercial transfection reagent TransIT ® (Mirus). Although unmodified H1M6 and H3M1 had high mRNA delivery efficiency in serum-free medium, no positive results were obtained in serum-containing medium, indicating that lipid modification plays an important role in enhancing the stability of the candidate polypeptide carrier@mRNA complex.

[0033] 2. Encapsulation and delivery of recombinant ARC protein to mRNA Recombinant preparation of ARC protein (the preparation method refers to CN 118576725A), and it was mixed with mRNA-Fluc at a mass ratio of 10:1 in PBS buffer for 10 min. Agarose gel electrophoresis showed that ARC could retard the electrophoretic migration of mRNA, confirming that ARC protein could interact with mRNA and form a complex. Compared with the degradation of naked mRNA by RNase, the ARC@mRNA complex could inhibit the degradation effect of RNase, confirming that ARC protein could encapsulate mRNA. However, cell delivery experiments showed that the recombinant ARC protein could not effectively deliver mRNA, and the fluorescence signal value had no statistical difference from the naked mRNA negative control (see Figure 5 ).

[0034] 3. Encapsulation and delivery of mRNA by cell-penetrating peptides TAT (SEQ ID NO.19: KGRKKRRQRRRPPQ), as a classical cell-penetrating peptide with an isoelectric point of 13.0, could also have electrostatic interaction with mRNA. Agarose gel electrophoresis experiments showed that the chemically synthesized TAT polypeptide (Sangon Biotech) could retard the electrophoretic migration of mRNA, indicating that it could form a complex with mRNA. However, cell delivery experiments showed that the TAT polypeptide could not effectively deliver mRNA, and the fluorescence signal value was even lower than that of the naked mRNA negative control (see Figure 6 ).

[0035] Example 2. Characterization of the properties of the biomimetic polypeptide carrier-mRNA complex (1) Characterization of the properties of the H3M1-5 biomimetic polypeptide carrier The molecular weight of H3M1-5 was detected by mass spectrometry (see Figure 7 ). According to the mass-to-charge ratio (978.70) of the highest molecular ion peak ([M + 3H] 3+ ) in the mass spectrometry diagram, the molecular weight of the polypeptide carrier was calculated to be 2933.10 Da, which was basically consistent with the theoretical value (2933.62 Da), indicating that the chemically synthesized polypeptide met the expectations. The biocompatibility of H3M1-5 was evaluated. 2×10 4 HeLa cells were seeded in 96-well plates and cultured overnight at 37 °C and 5% CO2. Different concentrations of H3M1-5 (0, 5, 10, 20, 40, 60, 80, and 100 μg / mL) were added to each well of the culture plate, and after 24 hours of culture, the CCK-8 kit was used for detection. The results showed that H3M1-5 had good biocompatibility, and no obvious cytotoxicity was produced even at a concentration as high as 100 μg / mL (see Figure 8 ).

[0036] (2) Characterization of the properties of the H3M1-5@mRNA complex H3M1-5 was mixed with mRNA-Fluc at N / P ratios of 2, 4, and 8 in 20 mM HEPES (pH 7.4) buffer. After interacting for 30 min, the particle size and potential were detected using dynamic light scattering (Malvern) (see Figure 9 ). The results showed that the diameter of the H3M1-5@mRNA complex decreased with the increase of the N / P ratio. The diameters at N / P ratios of 2, 4, and 8 were 180.4 nm, 97.9 nm, and 76.1 nm, respectively. The Zeta potential of the H3M1-5@mRNA complex showed a positive correlation with the N / P ratio. The potentials at N / P ratios of 2, 4, and 8 were +4.1 mV, +13.9 mV, and +21.5 mV, respectively, indicating that the encapsulation of H3M1-5 achieved a charge inversion of naked mRNA. The results of transmission electron microscopy (TEM) (see Figure 10 ) were consistent with those of dynamic light scattering. The H3M1-5@mRNA complex could form spherical nanoparticles with uniform morphology at different N / P ratios.

[0037] The RiboGreen kit (ThermoFisher) was used to detect the encapsulation effect of H3M1-5 on mRNA. The detection results showed that when the N / P ratio was above 4, H3M1-5 had a 100% encapsulation rate for mRNA (see Figure 11 ). The results of agarose gel electrophoresis showed (see Figure 12 ) that the interaction between the H3M1-5 peptide carrier and mRNA could hinder the migration of mRNA, and the H3M1-5@mRNA complex could protect the encapsulated mRNA from degradation by RNase. In addition, under the action of protease K, the H3M1-5@mRNA complex could dissociate and release the encapsulated mRNA, indicating that H3M1-5 could encapsulate mRNA and form a structure similar to the viral capsid, which could not only protect mRNA from degradation by nucleases but also help mediate the release of mRNA under the action of lysosomes / endosomes after the complex entered the cell and initiate the expression of target genes.

[0038] (3) Detection of the binding kinetics between the polypeptide carrier and mRNA The interaction between the polypeptide carrier and mRNA was quantitatively detected by biolayer interferometry (Gator). A sensor chip immobilized with streptavidin was used to capture in vitro transcribed and biotinylated mRNA-Fluc, which was immersed in polypeptide sample solutions at different concentration gradients, and the binding and dissociation curves were detected and the affinity constant was calculated (see Figure 13). The results showed that the initial polypeptide H3 did not exhibit obvious binding to mRNA-Fluc. After introducing amino acid mutations, the modified H3M1 could bind to mRNA-Fluc through electrostatic interactions. After further introducing C18 lipid modification, the affinity constant ( KD ) increased from 8.58×10 -9 M (H3M1@mRNA) to 3.86×10 -10 M (H3M1-5@mRNA). The above results indicate that compared with the initial H3 polypeptide derived from ARC, point mutation positive charge modification and lipid modification are of great significance for enhancing the interaction between the biomimetic polypeptide and mRNA.

[0039] Example 3. Cellular delivery of nucleic acids by biomimetic polypeptide carriers (1) Effect of salt ion concentration on the cellular delivery of H3M1-5@mRNA Based on a 20 mM HEPES (pH 7.4) buffer system, buffers containing different salt ion concentrations of 0, 25, 50, 100, and 200 mM NaCl were prepared. H3M1-5 and mRNA-Fluc were mixed at N / P ratios of 2, 4, and 8 in the above buffers. After interacting for 30 min, the HEK293T cells cultured overnight were transfected, and the fluorescence intensity was detected 24 hours later. The results showed that the bioluminescence intensity decreased with the increase in the salt ion strength of the solution. In the 20 mM HEPES (pH 7.4) buffer without NaCl, the delivery efficiency of the H3M1-5@mRNA-Fluc complex was the highest (see Figure 14 ).

[0040] (2) Delivery efficiency of H3M1-5@mRNA to different cell lines H3M1-5 and mRNA-Fluc were mixed at N / P ratios of 2, 4, and 8 in a 20 mM HEPES (pH 7.4) buffer. After interacting for 30 min, the cell lines of HeLa, HEK 293T, BHK 21, 3T3, and DC2.4 cultured overnight were transfected. The results showed that high efficient cellular delivery efficiency was exhibited in each cell line (see Figure 15 ), and the bioluminescence intensity was comparable to that of the positive control commercial transfection reagent TransIT ® (Mirus). It indicates that H3M1-5 can be used as a universal delivery vector for efficient mRNA delivery to different cells.

[0041] (3) Cellular delivery of H3M1-5@mRNA-GFP In vitro transcription was used to synthesize linearized mRNA encoding green fluorescent protein (mRNA-EGFP). In HEK 293T cells, the delivery efficiency of the H3M1-5@mRNA-EGFP complex at different N / P ratios was further verified. Flow cytometry results showed that when the N / P ratio was 4 and 8, 82.23% and 88.93% of the cells successfully expressed EGFP at 24 hours after transfection, respectively, and the positive cell ratio was higher than 79.67% of the commercial transfection reagent TransIT ® (Mirus) (see Figure 16 ).

[0042] (4) Cellular delivery of H3M1-5 to circular mRNA In HEK293T cells, the delivery effect of H3M1-5 on circular mRNA encoding luciferase (circRNA-Fluc, purchased from Novoprotein) was detected (see Figure 17 ). At different N / P ratios, the H3M1-5@circRNA-Fluc complex achieved high delivery efficiency, and the bioluminescence intensity was significantly higher than that of the naked mRNA negative control and close to that of the commercial transfection reagent TransIT ® (Mirus) positive control.

[0043] (5) Cellular delivery of H3M1-5 to plasmid DNA In HEK 293T cells, the delivery effects of H3M1-5 on plasmid DNA encoding luciferase (pDNA-Fluc) and green fluorescent protein (pDNA-GFP) (purchased from Hanheng Biotech) were detected respectively. At different N / P ratios, H3M1-5@ pDNA-Fluc could produce high-intensity bioluminescence signals, and there was no significant difference from the commercial transfection reagent TransIT ® (Mirus) positive control; while the delivery efficiency of H3M1-5@ pDNA-GFP increased with the increase of the mass ratio. When the N / P ratio was 2, 4, and 8, the positive cell percentages were 47.6%, 57.4%, and 63.7% respectively, reaching a delivery efficiency comparable to that of the commercial transfection reagent (TransIT ® ) positive control (see Figure 18 ).

[0044] (6) Efficient cellular gene editing by H3M1-5 delivered mRNA Based on the HEK293 cell line expressing unstable GFP (uGFP) (HEK293-uGFP, purchased from Gentarget), relying on the precise editing of the GFP gene by the CRISPR gene editing system, the efficiency of gene editing can be evaluated by detecting the change of green fluorescence signal in cells. The mRNA encoding the Cas9 gene (purchased from Novoprotein) was mixed with chemically synthesized sgRNAs (target sequences are 5'-CCGTCCAGCT CGACCAGGAT (SEQ ID NO.20) and 5'-CAAGACCCGCCACAACATCG (SEQ ID NO.21)) at a molar ratio of 1:1, and then mixed with H3M1-5 at N / P ratios (polypeptide / mRNA) of 2, 4, and 8 to form a complex. After 72 hours of transfection of HEK293-uGFP, the green fluorescence of cells was quantitatively detected by flow cytometry and fluorescence microscopy imaging. Compared with the negative control group transfected with only Cas9 without carrying sgRNA, the percentage of GFP-positive cells in the H3M1-5@Cas9 mRNA / sgRNA experimental group and the commercial transfection reagent (TransIT ® )positive control group showed a significant decrease, from 60% to about 26%, confirming that the efficient delivery of H3M1-5 to mRNA can be applied to cell gene editing (see Figure 19 ).

[0045] Example 4. In vivo delivery of mRNA by biomimetic polypeptide carrier and evaluation of immune protection (1)In vivo delivery of H3M1-5 to mRNA-Fluc 6-8-week-old female BALB / c mice (n = 3) were intradermally inoculated with 100 μL of H3M1-5@mRNA-Fluc at different N / P ratios (2, 4, 8) (5 μg mRNA / mouse). After 24 hours, the mice were intraperitoneally injected with luciferase substrate (30 mg / mL, 100 μL), and the fluorescence intensity in the mice was detected by in vivo imaging (PerkinElmer IVIS). The results showed that the in vivo expression efficiency of H3M1-5@mRNA-Fluc was the highest when the N / P ratio was 2 (see Figure 20 ).

[0046] 6-8-week-old female BALB / c mice (n = 3) were intradermally inoculated with 100 μL of H3M1-5@mRNA-Fluc at an N / P ratio of 2 (5 μg mRNA / mouse). In vivo bioluminescence detection was performed at 8, 24, 48, 96, 168, and 216 hours respectively. The results showed that the expression level could reach a relatively high level 8 hours after injection, and was significantly higher than that of the naked mRNA negative control group within the subsequent 7 days (see Figure 21 ).

[0047] (2)Application of H3M1-5 polypeptide carrier in infectious disease mRNA vaccines Using in vitro transcribed mRNA-M1R encoding the monkeypox virus M1R protein (synthesized with reference to CN117821482A) as a model mRNA molecule. In 20 mM HEPES (pH 7.4) buffer, H3M1-5 and mRNA-M1R were mixed at an N / P ratio of 2:1 to form the H3M1-5@mRNA-M1R complex. Female BALB / c mice aged 6-8 weeks (n = 5) were inoculated intradermally with three doses of high, medium, and low doses of H3M1-5@mRNA-M1R (100 μL / mouse), where the doses of mRNA were 5 μg / mouse, 10 μg / mouse, and 20 μg / mouse, respectively. The immunization schedule was three doses on days 0, 7, and 14. Mouse sera and splenocytes were collected 21 days after immunization, and humoral and cellular immunity were detected by ELISA and ELISpot.

[0048] ELISA results showed that the high-dose group of H3M1-5@20 μg mRNA-M1R could induce high levels of M1R-specific IgG antibodies 7 days after the last immunization, with an average titer reaching 10 3.99 , and the specific antibody levels among different dose groups showed a dose-dependent relationship, all significantly higher than those of the 20 mM HEPES negative control group. Vaccinia virus encoding luciferase (VACV-Luc, purchased from Yunzhou Biologics) was used to evaluate the neutralizing antibody levels in post-immunization sera. All different H3M1-5@mRNA-M1R dose groups could significantly activate the production of neutralizing antibodies, and the neutralizing antibody titer activated by H3M1-5@20 μg M1R mRNA was the highest, with an NT 50 reaching 10 2.26 , which was more than 8 times that of the negative control group (see Figure 22 ).

[0049] ELISpot was used to detect the cellular immune response induced by H3M1-5@20 μg mRNA-M1R. The splenocytes of immunized mice were stimulated with a full-length overlapping peptide library of M1R (synthesized by Sangon Biotech), and the number of interferon-γ (IFN-γ)-positive cells was quantitatively detected after 48 hours of culture. The results showed that the H3M1-5@mRNA-M1R vaccine significantly activated cellular immunity, inducing approximately 240 spots per million splenocytes, which was more than 5 times that of the negative control group (see Figure 23 ).

[0050] (3)Application of H3M1-5 polypeptide carrier in tumor mRNA vaccines A B16-OVA tumor model was constructed based on a mouse melanoma B16 cell line overexpressing chicken ovalbumin (OVA), and mRNA-OVA encoding the OVA antigen was synthesized to evaluate the anti-tumor effect of the mRNA vaccine. In 20 mM HEPES (pH 7.4) buffer, H3M1-5 and mRNA-OVA were mixed at an N / P ratio of 2:1 to form the H3M1-5@mRNA-M1R complex. Female C57BL / 6 mice aged 6-8 weeks were inoculated intradermally with three doses of the vaccine on days 0, 7, and 14, and were given 20 mM HEPES, LNP@5 μg mRNA-OVA, H3M1-5@5 μg mRNA-OVA, and H3M1-5@10 μg mRNA-OVA, respectively. Among them, LNP@5 μg mRNA-OVA was the liposome nanoparticle positive control group (the preparation method of LNP refers to CN117821482A), and PBS was the negative control group. Seven days after the last immunization, mouse sera were collected to detect the OVA-specific antibody titer, and at the same time, the spleens and lymph nodes of the mice were collected to evaluate the cellular immune level.

[0051] The ELISA results showed (see Figure 24 ), the OVA-specific antibody titers induced by H3M1-5@5 μg mRNA-OVA and H3M1-5@10 μg mRNA-OVA were 10 2.65 and 10 3.37 , slightly lower than the antibody titer of LNP@5 μg OVA mRNA (10 4.24 ). The ELISpot results showed (see Figure 25 ), after in vitro stimulation of splenocytes with the short peptide SIINFEKL, H3M1-5@mRNA-OVA significantly activated cellular immunity. Although it was lower than the LNP positive control, H3M1-5@10 μg mRNA-OVA could still induce a relatively high level of cellular immunity, generating approximately 650 IFN-γ positive spots (per million splenocytes), which was more than 30 times that of the negative control group.

[0052] In the B16-OVA melanoma treatment model, female C57BL / 6 mice aged 6-8 weeks were subcutaneously inoculated with 3×10 520 mM HEPES, LNP@5 μg mRNA-OVA, H3M1-5@5 μg mRNA-OVA, and H3M1-5@10 μg mRNA-OVA were respectively injected intradermally into B16-OVA melanoma cells. After 7 days, the immunization process was three intradermal inoculations at days 0, 7, and 14. The changes in the tumor volume of mice were measured with a vernier caliper. Compared with the negative control group, H3M1-5@10 μg mRNA-OVA significantly inhibited tumor growth, and there was no significant difference in the tumor suppression effect compared with the LNP positive control group. The negative control group and the experimental group of mice were dissected on days 20 and 23 respectively, and the tumor weights were measured. The tumor weights and sizes of the H3M1-5@10 μg mRNA-OVA experimental group and the LNP@5 μg mRNA-OVA positive control group were significantly smaller than those of the negative control (see Figure 26 ).

[0053] (4)Component splicing enhances the immunogenicity of polypeptide biomimetic vector mRNA vaccine Polypeptide carriers have the advantage of modular splicing of delivery elements, which can further improve the delivery efficiency of candidate polypeptides. Peptide 22A (sequence PVLDLFRELLNELLEALKQKLK (SEQ ID NO.22)) is an apolipoprotein mimetic polypeptide composed of 22 amino acids, with certain lymph node targeting and adjuvant effects. Peptide 22A was fused to the N-terminus of H3M1-5, and the 22A-H3M1-5 fusion peptide was chemically synthesized (synthesized by Sangon Biotech). In 20 mM HEPES (pH 7.4) buffer, 22A-H3M1-5 and mRNA-OVA were mixed at an N / P ratio of 2:1 to form the 22A-H3M1-5@mRNA-M1R complex.

[0054] In BALB / c mice, 20 mM HEPES, H3M1-5@20 μg mRNA-M1R, and 22A-H3M1-5@20 μg mRNA-M1R were inoculated intradermally at days 0, 7, and 14. Seven days after the last immunization, ELISA results showed that the 22A-H3M1-5 immunized group (average titer 10 5.61 exhibited a stronger antibody response compared with the H3M1-5 group (average titer 10 4.85 ), and the titer of M1R-specific IgG antibody increased by more than 5 times (see Figure 27 ).

[0055] Evaluate the immunoprotection of the candidate monkeypox mRNA vaccine in a lethal mousepox virus challenge model (refer to CN117821482A). Fourteen days after the last immunization, 50 PFU of mousepox virus was injected intraperitoneally. Compared with all the mice in the control group dying within 6 days, the survival rates of the immunized mice inoculated with H3M1-5@20 μg mRNA-M1R and 22A-H3M1-5@20 μg mRNA-M1R reached 80% and 100% respectively, indicating that both peptide-based carrier vaccines had high immunoprotection (see Figure 28 ).

[0056] Example 5. Safety evaluation of biomimetic polypeptide carrier mRNA vaccine Previous studies have shown that LNP has high pro-inflammatory activity, which has an adverse effect on the safety of LNP-mRNA vaccines. Compare and evaluate the safety of LNP and peptide-based carrier mRNA in a BALB / c mouse model. Intradermal inoculation of naked mRNA-OVA control group, LNP@5 μg mRNA-OVA, H3M1-5@10 μg mRNA-OVA, and 22A-H3M1-5@10 μg mRNA-OVA was performed as a single dose. Mouse serum was collected 4 hours later and the content of inflammatory cytokines was detected. The results showed that the levels of cytokines such as IL-6, IL-13, KC, and MCP-1 in the serum of the LNP immunized group were significantly higher than those of the control group. For example, the level of IL-6 was more than 10 times that of the control group; while there was no significant difference between the H3M1-5 and 22A-H3M1-5 immunized groups and the control group, and no obvious inflammatory cytokines were activated (see Figure 29 ).

[0057] In addition, LNP in vivo delivery has liver tropism and is prone to accumulate in large amounts in the liver, with potential safety risks. Single-dose intradermal immunization of LNP@1 μg mRNA-Fluc and 22A-H3M1-5@5 μg mRNA-Fluc, and in vivo or tissue and organ (heart, liver, spleen, lung, and kidney) imaging of mice was performed 24 hours later. The results showed that in addition to signal enhancement at the injection site, LNP@mRNA-Fluc also had significant accumulation in the liver; while 22A-H3M1-5@mRNA-Fluc only aggregated at the injection site and did not migrate to other organs (see Figure 30 ).

[0058] Furthermore, the blood biochemical indexes after multiple immunizations were detected. BALB / c mice were inoculated intradermally on days 0, 7, and 14, and each mouse was immunized with 20 mM HEPES, H3M1-5@20 μg mRNA-M1R, 22A-H3M1-5@20 μg mRNA-M1R, and LNP@5 μg mRNA-M1R. Serum was collected on day 21 for blood biochemical detection. The results showed that there were no differences in the indexes of ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), and UREA (urea) between the immunized groups and the control group (see Figure 31 ).

Claims

1. A polypeptide variant based on the ARC protein, characterized in that, The described polypeptide variant based on the ARC protein is a variant designed based on the spatial conformation, positive charge modification by amino acid point mutation, and side chain modification of a lipid group or lipid derivative. The spatial conformation is an α-helix, and the goal of the positive charge modification by amino acid point mutation is to increase the isoelectric point to above 10.

0.

2. The polypeptide variant based on the ARC protein according to claim 1, wherein The amino acid sequence of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.

14.

3. The polypeptide variant based on the ARC protein according to claim 2, wherein, The lipid group or lipid derivative is octadecyl or cholesterol.

4. The polypeptide variant based on the ARC protein according to claim 2, wherein The position where the side chain modification of the lipid group or lipid derivative occurs is at the N-terminus, C-terminus or middle position of the polypeptide variant based on the ARC protein. When the position where the side chain modification of the lipid group or lipid derivative occurs is at the C-terminus of the polypeptide variant based on the ARC protein, lysine for providing the side chain modification position of the lipid group or lipid derivative is further provided at the C-terminus.

5. The polypeptide variant based on the ARC protein according to claim 4, wherein The sequence of the side chain modification of the lipid group of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.

14. The lipid group is octadecyl, and the modification position is the C-terminus.

6. The polypeptide variant based on the ARC protein according to claim 4, wherein A linking element is provided between the polypeptide variant based on the ARC protein and the lipid group or lipid derivative.

7. The polypeptide variant based on the ARC protein according to claim 6, wherein The linking element is GSG and / or polyethylene glycol.

8. The polypeptide variant based on the ARC protein according to claim 7, wherein The sequence of the lipid modification of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.

7. The lipid group is octadecyl, the modification position is the C-terminus, and the linking element is GSG-K-PEG8.

9. The polypeptide variant based on the ARC protein according to claim 7, characterized in that, The sequence of the side chain modification of the lipid group of the polypeptide variant based on the ARC protein is as shown in SEQ ID NO.

14. The lipid group is octadecyl, the modification position is the N-terminus or C-terminus, and the linking element is GSG.

10. The polypeptide variant based on the ARC protein according to claim 2, characterized in that, The polypeptide variant based on the ARC protein is further fused with a polypeptide having a targeting function and / or adjuvant effect.

11. The polypeptide variant based on the ARC protein according to claim 10, characterized in that, The sequence of the polypeptide having a targeting function and / or adjuvant effect is as shown in SEQ ID NO.

22.

12. Use of the polypeptide variant based on the ARC protein according to any one of claims 2-11 as a nucleic acid delivery vector.

13. The application according to claim 12, wherein The use is for preparing a gene editing vector or preparing an mRNA vaccine.

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