A modified polypeptide based on arc protein and its use as a nucleic acid delivery vehicle

Abstract

The present application provides a polypeptide variant based on ARC protein and its application 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 lipid base or lipid base derivative. The spatial conformation is alpha helix, and the target of the point mutation is to raise the isoelectric point to above 10.0. The polypeptide variant has excellent nucleic acid affinity constant, can wrap mRNA or DNA and form a structure similar to a viral capsid, can protect mRNA from degradation by nucleases, and also helps to mediate the release of mRNA or DNA after the complex enters the cell under the action of lysosomes / endosomes, can activate effective humoral and cellular immune responses in vivo, has significant immunoprotection against mousepox challenge and melanoma, and has good biocompatibility, and therefore can be prepared into a new nucleic acid delivery carrier based on biomimetic polypeptide.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel nucleic acid delivery carrier and its application in mRNA vaccines and gene therapy, belonging to the technical field of biological medicine. BACKGROUND

[0002] mRNA vaccines have become an important direction of new vaccine research and development due to their rapid synthesis, strong immune activation and other characteristics. Due to the degradation and difficulty of mRNA molecules in crossing the membrane, the carrier delivery system is the key core technology of mRNA vaccines. At present, Lipid Nanoparticle (LNP) is the most widely used mRNA vaccine delivery system, which is mainly composed of four kinds of lipids, including ionizable lipids or cationic lipids, cholesterol, auxiliary phospholipids and PEGylated lipids. By microfluidic technology, the four lipids dissolved in the ethanol phase are mixed with the mRNA molecules in the water phase in proportion to realize the encapsulation of mRNA and form uniform nanoparticles. The mRNA vaccine based on LNP can activate a strong immune response, but compared with other technology platform vaccines, clinical data show that the marketed COVID-19 mRNA vaccines have a higher adverse reaction rate, including fever rate, myocarditis, autoimmune diseases, etc. At present, the LNP is optimized mainly by adjusting the LNP formula, developing new ionizable lipids, etc., but it does not fundamentally solve the inherent bottlenecks of the LNP system.

[0003] The development of a new concept of safe and efficient new mRNA delivery carriers has become an important direction of mRNA innovation technology development. Based on the self-assembly between proteins and nucleic acids, the protein coat of the virus can encapsulate nucleic acids and form natural nanoparticles with high cell infection capacity. In addition, endogenous retrovirus-like proteins such as PEG10, ARC, etc. in mammals can also encapsulate their own mRNA and play specific functions in the body through extracellular vesicles. It has been reported that based on the similar virus carrier or pseudovirus packaging method, virus-like particles that can deliver mRNA are prepared by transfecting cells with lentiviral plasmids, but these strategies are complicated and cumbersome in cell culture, separation and purification, which brings great challenges to quality control.

[0004] The purpose of the present application is based on the virus biomimetic concept of protein coat encapsulating nucleic acids, using a technical route different from the LNP-dependent multi-component high molecular lipids and the virus-like packaging-dependent cell transfection, developing a new type of single-component carrier based on peptides in a controllable self-assembly manner in vitro, realizing the efficient encapsulation and delivery of mRNA and applying it to the research and development of new mRNA vaccines and drugs. SUMMARY

[0005] Based on the above purpose, the present application firstly provides a polypeptide variant based on ARC protein, which is a variant based on spatial conformation design, positive electric reconstruction of amino acid point mutation and side chain modification, the spatial conformation is alpha helix, the target of positive electric reconstruction of amino acid point mutation is to improve the isoelectric point to above 10.0, and the polypeptide variant based on ARC protein is further subjected to side chain modification of lipid group or lipid group derivative.

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

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

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

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

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

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

[0012] In a specific embodiment of the present application, the sequence of the side chain modification of the lipid group of the ARC protein-based polypeptide variant is shown in SEQ ID NO. 7, the lipid group is octadecyl, the modification position is the C-terminal, and the connecting element is GSG-K-PEG8. In the present application, the ARC protein-based polypeptide variant is named “H1M6-7”.

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

[0014] In a specific embodiment of the present application, when the substitution position is the N-terminal, the substituent is C18, and the connecting element is GSG, the ARC protein-based polypeptide variant is named “H3M1-2”.

[0015] In a specific embodiment of the present application, when the substitution position is the C-terminal, the substituent is C18, and the connecting element is GSG, the ARC protein-based polypeptide variant is named “H3M1-5”.

[0016] In a preferred embodiment of the present application, the ARC protein-based polypeptide variant 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 shown in SEQ ID NO. 22. In the present application, the polypeptide having a sequence shown in SEQ ID NO. 22 is named 22A.

[0018] In a specific embodiment of the present application, the polypeptide 22A is fused to the N-terminal of the ARC protein-based polypeptide variant “H3M1-5”, and the fused polypeptide is named “22A-H3M1-5 fusion peptide”.

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

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

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

[0022] In one specific embodiment of the present invention, the application is the preparation of an mRNA vaccine.

[0023] This invention provides a polypeptide variant based on the ARC protein. The variant originates from a polypeptide with an α-helix conformation within the ARC protein. After introducing positively charged amino acid point mutations and side-chain modifications with lipid groups or lipid derivatives, the affinity constant for nucleic acids (…) is… KD The biomimetic peptide delivery vector has been significantly improved, capable of encapsulating mRNA or DNA to form a viral capsid-like structure. This protects mRNA from nuclease degradation and facilitates the release of mRNA and initiation of target gene expression via lysosomes / endosomes after the complex enters the cell. It also exhibits good biocompatibility, making it suitable for development into novel nucleic acid delivery vectors based on biomimetic peptides. These vectors can achieve highly efficient encapsulation and delivery of in vitro transcribed mRNA and plasmid DNA through self-assembly. The single-component biomimetic peptide delivery vector provided by this invention can easily form structurally stable, uniformly sized nanocomplexes with nucleic acids through simple mixing, resulting in highly efficient delivery at both cellular and in vivo levels. In mouse models, delivery of mRNA vaccines encoding model antigens such as monkeypox virus M1R and OVA effectively activates humoral and cellular immune responses and provides significant immunoprotection against mousepox challenge and melanoma. Compared to four-component liposome nanoparticles (LNPs) with strong inflammatory activity and liver enrichment, this single-component biomimetic peptide-delivered mRNA vaccine or gene editing vector expresses its target gene only at the injection site and does not activate the production of significant inflammatory cytokines. Furthermore, the peptide vector has the advantage of modular splicing of delivery elements, allowing it to fuse with other vector peptides to further improve delivery efficiency. The ARC protein-based peptide variants provided by this invention hold promise for offering a safer, more effective, and easier-to-use new strategy for the development of novel nucleic acid vaccines and gene therapy drugs. Attached Figure Description

[0024] Figure 1 The technical roadmap for the design, screening, and optimization of the biomimetic carrier of this invention;

[0025] Figure 2 Alphafold3 predicts the spatial conformation of candidate peptides;

[0026] Figure 3 First-round candidate peptide vector mRNA cell delivery efficiency assay;

[0027] Figure 4 Second round of candidate peptide vector mRNA cell delivery efficiency assay;

[0028] Figure 5 . Detection of mRNA encapsulation and cell delivery of ARC recombinant protein;

[0029] Figure 6 . Detection of mRNA encapsulation and cell delivery of TAT polypeptide;

[0030] Figure 7 . Mass spectrometry detection of H3M1-5 molecular weight;

[0031] Figure 8 . H3M1-5 cytotoxicity detection;

[0032] Figure 9 . H3M1-5 and mRNA-Fluc self-assembled complex particle size and potential detection;

[0033] Figure 10 . Transmission electron microscopy detection of H3M1-5 and mRNA-Fluc self-assembled complex;

[0034] Figure 11 . H3M1-5@mRNA-Fluc complex encapsulation rate detection;

[0035] Figure 12 . H3M1-5@mRNA-Fluc complex gel retardation experiment;

[0036] Figure 13 . Detection of polypeptide carrier and mRNA binding kinetics;

[0037] Figure 14 . Detection of cell delivery efficiency of H3M1-5@mRNA-Fluc under different salt ion concentrations;

[0038] Figure 15 . Detection of delivery efficiency of H3M1-5@mRNA-Fluc in different cell lines;

[0039] Figure 16 . Detection of cell delivery efficiency of H3M1-5@mRNA-GFP;

[0040] Figure 17 . Detection of cell delivery efficiency of H3M1-5 to circular mRNA;

[0041] Figure 18 . Detection of cell delivery efficiency of H3M1-5 to plasmid DNA;

[0042] Figure 19 . Detection of cell gene editing efficiency of H3M1-5 delivered CRSPR system;

[0043] Figure 20 . Detection of in vivo delivery efficiency of H3M1-5@mRNA-Fluc at different N / P ratios;

[0044] Figure 21 . Detection of in vivo delivery efficiency of H3M1-5@mRNA-Fluc at different time points;

[0045] Figure 22 . Detection of antibody response of H3M1-5@mRNA-M1R monkeypox vaccine;

[0046] Figure 23 . Detection of cellular immune response of H3M1-5@mRNA-M1R monkeypox vaccine;

[0047] Figure 24 . Detection of antibody response of H3M1-5@mRNA-OVA tumor vaccine;

[0048] Figure 25 . Detection of cellular response of H3M1-5@mRNA-OVA tumor vaccine;

[0049] Figure 26 . Evaluation of anti-tumor effect of H3M1-5@mRNA-OVA in melanoma model;

[0050] Figure 27 . Detection of antibody response of 22A-H3M1-5@mRNA-M1R monkeypox vaccine;

[0051] Figure 28 . Detection of lethal challenge protection of 22A-H3M1-5@mRNA-M1R monkeypox vaccine;

[0052] Figure 29 . Detection of cytokine levels after immunization of LNP and polypeptide carrier mRNA-OVA vaccine;

[0053] Figure 30 . Detection of in vivo biodistribution after delivery of mRNA-Fluc by LNP and polypeptide carrier;

[0054] Figure 31 . Blood biochemical detection after delivery of mRNA-M1R by LNP and polypeptide carrier. DETAILED DESCRIPTION

[0055] The present application will be further described with reference to the following examples. The advantages and characteristics of the present application will become more apparent with the description. However, these examples are only exemplary and do not constitute any limitation to the protection scope defined by the claims of the present application.

[0056] Example 1. Design and screening optimization of biomimetic carrier

[0057] Figure 1 The technical roadmap of the design and screening optimization of the biomimetic carrier of the present application is given. First, the polypeptide carrier design based on alpha helix and isoelectric point is carried out, the candidate ARC protein-based polypeptide variant is obtained, then the linear mRNA encoding luciferase is packaged to form a virus-like capsid structure, then the first round of screening of the candidate polypeptide based on the fluorescence intensity is carried out, then the first round of screening of the candidate polypeptide is carried out and the lipid-based modification is carried out, and the second round of screening of the candidate polypeptide based on the fluorescence intensity is carried out, and finally the ARC protein-based polypeptide variant is obtained.

[0058] 1. Design and screening of polypeptide variants of ARC protein

[0059] (1) Polypeptide carrier design based on alpha helix and isoelectric point

[0060] The candidate protein structure is analyzed by Alphafold3 and other auxiliary tools, taking ARC protein as an example, polypeptides with alpha helix conformation and isoelectric point greater than 7.0 are selected, which are ARC 99-126 (H1), ARC 249-257 (H2), ARC 297-313 (H3) and ARC 329-339 (H4). On the basis of H1-H4 polypeptides, the amino acid sequence is restructured in combination with electrostaticity and amphiphilicity, the isoelectric point of the candidate polypeptide is improved to more than 10.0 on the basis of maintaining the alpha helix conformation, so as to enhance the interaction with mRNA. The design parameters include: j, the number of amino acid point mutations; k, the tandem mode of repeated sequences (forward or reverse), etc. A total of 18 candidate polypeptides are designed and chemically synthesized (Shenguo Bioengineering). Alphafold3 prediction shows that most of the candidate polypeptides can maintain the alpha helix (see Figure 2 ), and the sequence restructuring does not cause significant changes to the basic conformation of the original polypeptide.

[0061] Table 1. First round of candidate polypeptide design

[0062]

[0063] (2) Screening of the first round of candidate polypeptide carriers

[0064] In a cell model, the delivery efficiency of the first round of candidate polypeptides to mRNA was detected. DC2.4 cells (20,000 per well, 100 μL) were cultured in a 96-well plate overnight (37°C, 5% CO2) with DMEM medium containing 10% fetal bovine serum (FBS). The candidate polypeptides were mixed with linearized mRNA encoding luciferase (mRNA-Fluc, purchased from Novozyme) at a N / P ratio of 20:1 in 20 mM HEPES (pH 7.4) buffer for 30 min. Before transfection of the cells, the culture medium was replaced with Opti-MEM reduced serum medium, and 125 ng of mRNA was added to each well (n=3). After 4 hours of transfection, the cells were replaced with 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 luciferase 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 efficiency of H1M6 (SEQ ID NO. 7), H3M1 (SEQ ID NO. 14), and H1M5 (SEQ ID NO. 6) to mRNA was significantly higher than that of the negative control, while the original polypeptides H1-H4 derived from ARC did not produce positive results (see Figure 3 ).

[0065] (3) Optimization of candidate polypeptide carriers based on lipid-based modification

[0066] To further enhance the hydrophobicity of the candidate polypeptides and their interaction with the cell membrane, thereby improving the cell delivery efficiency of mRNA, the candidate polypeptides H1M6 and H3M1 selected in the first round were modified with lipid-based modification. 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-terminal, C-terminal, or middle position of the candidate polypeptide; and l, whether there is a linker such as GSG (glycine-serine-glycine) or polyethylene glycol (PEG) between the candidate polypeptide and the modified lipid-based. Eighteen candidate polypeptides were designed and chemically synthesized (Shenguo Bioengineering) according to the optimization design in Table 2.

[0067] Table 2. Design of second round of candidate polypeptides

[0068]

[0069] (4) Screening of the second round of candidate polypeptide carriers

[0070] In a serum-transfected cell model, the mRNA delivery efficiency of the second-round candidate peptides was assessed. DC2.4 cells (20,000 cells / well, 100 μL) were cultured overnight in 96-well plates (37°C, 5% CO2) in DMEM containing 10% fetal bovine serum. Candidate peptides and mRNA-luc were mixed at an N / P ratio of 2, 4, and 8 in 20 mM HEPES (pH 7.4) buffer. After 30 min of interaction, cells were transfected (n=3, 125 ng mRNA / well). Fluorescein substrate was added to the cell culture plates 24 hours later to detect fluorescence intensity. Results (see...) Figure 4 The results showed that, under conditions with 10% serum, H1M6-7, H3M1-2, H3M1-4, and H3M1-5 all exhibited good mRNA delivery efficiency, with H3M1-5 showing the highest mRNA delivery efficiency and a fluorescence intensity reaching 8 × 10⁻⁶. 7 When the RLU level was above 1, and 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 showed high mRNA delivery efficiency in serum-free medium, they did not produce positive results in serum-containing medium, indicating that lipid modification plays an important role in improving the stability of candidate peptide carrier@mRNA complexes.

[0071] 2. Encapsulation and delivery of mRNA by recombinant ARC protein

[0072] Recombinant ARC protein was prepared (preparation method according to CN 118576725A) and mixed with mRNA-Fluc at a mass ratio of 10:1 in PBS buffer for 10 min. Agarose gel electrophoresis showed that ARC could block the electrophoretic migration of mRNA, confirming that ARC protein can interact with mRNA and form a complex. Compared with the degradation of naked mRNA by RNases, the ARC@mRNA complex can inhibit the degradation of RNases, confirming that ARC protein can encapsulate mRNA. However, cell delivery experiments showed that recombinant ARC protein could not effectively deliver mRNA, and the fluorescence signal value was not statistically different from that of the naked mRNA negative control (see [link to documentation]). Figure 5 ).

[0073] 3. Encapsulation and delivery of mRNA by cell-penetrating peptides

[0074] TAT (SEQ ID NO. 19: KGRKKRRQRRRPPQ) as a classic cell penetrating peptide, isoelectric point is 13.0, can also interact with mRNA electrostatically. Agarose gel electrophoresis experiments show that the chemically synthesized TAT polypeptide (Shanghai Shenergy Biotech) can retard the electrophoretic migration of mRNA, indicating that it can form a complex with mRNA, however, cell delivery experiments show that TAT polypeptide cannot effectively deliver mRNA, and the fluorescence signal value is even lower than that of the naked mRNA negative control (see Figure 6 ).

[0075] Example 2. Characterization of the properties of the biomimetic polypeptide carrier-mRNA complex

[0076] (1) Characterization of the properties of H3M1-5 biomimetic polypeptide carrier

[0077] The molecular weight of H3M1-5 was detected by mass spectrometry (see Figure 7 ), and the mass-to-charge ratio of the highest molecular ion peak ([M+3H] 3+ ) in the mass spectrum was 978.70, indicating that the molecular weight of the polypeptide carrier was 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, 2x10 4 HeLa cells were seeded in a 96-well plate and incubated at 37°C, 5% CO2 overnight. 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 the CCK-8 kit was used for detection after 24 hours of culture. The results showed that H3M1-5 had good biocompatibility, and even at a concentration as high as 100 μg / mL, no significant cytotoxicity was observed (see Figure 8 ).

[0078] (2) Characterization of the properties of H3M1-5@mRNA complex

[0079] H3M1-5 and mRNA-Fluc were mixed at N / P ratios of 2, 4 and 8 in 20 mM HEPES (pH 7.4) buffer, and the particle size and zeta potential were detected by dynamic light scattering (Malvern) after 30 min of interaction (see Figure 9). The results show that the diameter of H3M1-5@mRNA complex decreases with the increase of N / P ratio, and the diameters at N / P ratios of 2, 4, and 8 are 180.4 nm, 97.9 nm, and 76.1 nm, respectively. The zeta potential of H3M1-5@mRNA complex is positively correlated with the N / P ratio, and the potentials at N / P ratios of 2, 4, and 8 are +4.1 mV, +13.9 mV, and +21.5 mV, respectively, indicating that the charge reversal of naked mRNA is achieved by H3M1-5 encapsulation. Transmission electron microscopy (TEM) results (see Figure 10 ) are consistent with the results of dynamic light scattering, and H3M1-5@mRNA complex can form spherical nanoparticles with uniform morphology at different N / P ratios.

[0080] The RiboGreen kit (ThermoFisher) is used to detect the encapsulation effect of H3M1-5 on mRNA. The detection results show that when the N / P ratio is above 4, H3M1-5 has a 100% encapsulation rate on mRNA (see Figure 11 ). The results of agarose gel electrophoresis show (see Figure 12 ) that the interaction between H3M1-5 peptide-based carrier and mRNA can hinder the migration of mRNA, and H3M1-5@mRNA complex can protect the encapsulated mRNA from degradation by RNase. In addition, under the action of protease K (Protease K), H3M1-5@mRNA complex can dissociate and release the encapsulated mRNA, indicating that H3M1-5 can encapsulate mRNA and form a structure similar to a viral capsid, which can protect mRNA from degradation by nucleases and also help to release mRNA and initiate target gene expression after the complex enters the cell under the action of lysosomes / endosomes.

[0081] (3) Detection of the binding kinetics of polypeptide carrier and mRNA

[0082] The interaction between polypeptide carrier and mRNA is quantitatively detected by bio-layer interferometry (Gator). The sensor chip with immobilized streptavidin is used to capture in vitro transcribed and synthesized biotin-modified mRNA-Fluc, and the polypeptide sample solution at different concentration gradients is immersed, the binding and dissociation curves are detected, and the affinity constant is calculated (see Figure 13 ). The results show that the initial polypeptide H3 does not exhibit obvious binding with mRNA-Fluc, and after introducing amino acid mutations, the modified H3M1 can bind with mRNA-Fluc through electrostatic interaction. After further introducing C18 lipid modification, the affinity constant ( KD ) is increased from 8.58 x 10 -9 M (H3M1@mRNA) to 3.86 x 10 -10M (H3M1-5@mRNA). The above results show that the positive charge engineering and lipid-based modification are important for enhancing the interaction between the biomimetic peptide and mRNA compared to the initial ARC-derived H3 polypeptide.

[0083] Example 3. Cell delivery of nucleic acids by biomimetic peptide carriers

[0084] (1) Effect of salt ion concentration on H3M1-5@mRNA cell delivery

[0085] Based on a 20 mM HEPES (pH 7.4) buffer system, buffers containing 0, 25, 50, 100, 200 mM NaCl at different salt ion concentrations were prepared. H3M1-5 and mRNA-Fluc were mixed at N / P ratios of 2, 4, 8 in the above buffers, and after 30 min of interaction, the overnight cultured HEK293T cells were transfected, and the fluorescence intensity was detected after 24 hours. The results show that the bioluminescence intensity decreases with increasing salt ion strength of the solution, and in the 20 mM HEPES (pH 7.4) buffer without NaCl, the delivery efficiency of the H3M1-5@mRNA-Fluc complex is the highest (see Figure 14 ).

[0086] (2) Delivery efficiency of H3M1-5@mRNA to different cell lines

[0087] H3M1-5 and mRNA-Fluc were mixed at N / P ratios of 2, 4, 8 in 20 mM HEPES (pH 7.4) buffer, and after 30 min of interaction, the overnight cultured HeLa, HEK 293T, BHK 21, 3T3, DC2.4 cell lines were transfected. The results show that high cell delivery efficiency is exhibited in each cell line (see Figure 15 ), and the bioluminescence intensity is comparable to that of the positive control commercial transfection reagent TransIT ® (Mirus). This indicates that H3M1-5 can be used as a universal delivery carrier for efficient mRNA delivery in different cells.

[0088] (3) Cell delivery of H3M1-5@mRNA-GFP

[0089] Linearized mRNA (mRNA-EGFP) encoding green fluorescent protein was synthesized by in vitro transcription, and the delivery efficiency of H3M1-5@mRNA-EGFP complex at different N / P ratios was further verified in HEK 293T cells. The flow cytometry results show that at N / P ratios of 4 and 8, 82.23% and 88.93% of cells successfully expressed EGFP 24 hours after transfection, respectively, and the positive cell ratio is higher than that of the positive control commercial transfection reagent TransIT® (Mirus) at 79.67% (see Figure 16 ).

[0090] (4) Cell delivery of H3M1-5 to circular mRNA

[0091] In HEK293T cells, the delivery effect of H3M1-5 to circular mRNA encoding luciferase (circRNA-Fluc, purchased from Coastal Pro) was detected (see Figure 17 ), and the H3M1-5@ circRNA-Fluc complex reached a high delivery efficiency at different N / P ratios, and the bioluminescence intensity was significantly higher than that of the naked mRNA negative control, and close to the commercial transfection reagent TransIT ® (Mirus) positive control.

[0092] (5) Cell delivery of H3M1-5 to plasmid DNA

[0093] In HEK 293T cells, the delivery effect of H3M1-5 to plasmid DNA (purchased from Hanheng Biotechnology) encoding luciferase (pDNA-Fluc) and green fluorescent protein (pDNA-GFP) was detected. At different N / P ratios, H3M1-5@ pDNA-Fluc can produce high-intensity bioluminescence signals, and there is no obvious difference with the commercial transfection reagent TransIT ® (Mirus) positive control; while the delivery efficiency of H3M1-5@ pDNA-GFP increases with the increase of mass ratio, and the percentage of positive cells at N / P ratios of 2, 4 and 8 is 47.6%, 57.4% and 63.7% respectively, reaching the delivery efficiency comparable to that of the commercial transfection reagent (TransIT ® ) positive control (see Figure 18 ).

[0094] (6) H3M1-5 delivers mRNA to achieve efficient cell gene editing

[0095] Based on the HEK293 cell line expressing unstable GFP (Unstable GFP, uGFP) (HEK293-uGFP, purchased from Gentarget), the precise editing of the GFP gene by the CRISPR gene editing system can evaluate the efficiency of gene editing by detecting the changes of green fluorescence signal of cells. The mRNA encoding Cas9 gene (purchased from Coast Protein) was mixed with chemically synthesized sgRNA (targeting 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 an N / P ratio (polypeptide / mRNA) of 2, 4, 8 to form a complex. After transfection of HEK293-uGFP for 72 hours, the green fluorescence of cells was quantitatively detected by flow cytometry and fluorescence microscopy imaging. Compared with the negative control group transfected with Cas9 without carrying sgRNA, the GFP positive cell percentage of the H3M1-5@Cas9 mRNA / sgRNA experimental group and the commercial transfection reagent (TransIT ® ) positive control group decreased significantly from 60% to about 26%, which confirmed that the efficient delivery of mRNA by H3M1-5 could be applied to cell gene editing (see Figure 19 ).

[0096] Example 4. In vivo delivery of biomimetic polypeptide carriers for mRNA and evaluation of immune protection

[0097] (1) In vivo delivery of H3M1-5 for mRNA-Fluc

[0098] 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 per mouse), and 24 hours later, the mice were injected intraperitoneally with luciferase substrate (30 mg / mL, 100 μL). The in vivo fluorescence intensity of the mice was detected by live imaging (PerkinElmer IVIS), and the results showed that the in vivo expression efficiency of H3M1-5@mRNA-Fluc was the highest at an N / P ratio of 2 (see Figure 20 ).

[0099] H3M1-5@mRNA-Fluc at N / P ratio of 2 was used for intradermal injection (5 μg mRNA per mouse) in 6-8 week old female BALB / c mice (n=3), and the in vivo bioluminescence detection was performed at 8, 24, 48, 96, 168 and 216 hours, respectively. The results showed that the expression level reached a high level at 8 hours after injection, and was significantly higher than that of the naked mRNA negative control group in the subsequent 7 days (see Figure 21 ).

[0100] (2) Application of H3M1-5 polypeptide vector in infectious disease mRNA vaccine

[0101] The in vitro transcription mRNA-M1R (synthetic reference CN117821482A) encoding the monkeypox virus M1R protein was used as a model mRNA molecule. H3M1-5 and mRNA-M1R were mixed at an N / P ratio of 2:1 in 20 mM HEPES (pH 7.4) buffer to form H3M1-5@mRNA-M1R complexes. 6-8 week old female BALB / c mice (n=5) were intradermally injected with H3M1-5@mRNA-M1R at high, medium and low doses (100 μL per mouse), with the mRNA doses being 5 μg per mouse, 10 μg per mouse and 20 μg per mouse, respectively. The immunization program was three doses of injection on days 0, 7 and 14, and the mouse serum and spleen lymphocytes were collected 21 days after immunization for detection of humoral immunity and cellular immunity by ELISA and ELISpot.

[0102] The ELISA results showed that the H3M1-5@20 μg mRNA-M1R high-dose group could induce high levels of M1R-specific IgG antibodies 7 days after the last immunization, with an average titer of 10 3.99 , and the specific antibody levels in different dose groups showed a dose-dependent manner, which were significantly higher than those of the 20 mM HEPES negative control group. The vaccinia virus encoding luciferase (VACV-Luc, purchased from Yunzhou Biotechnology) was used to evaluate the neutralizing antibody levels in the post-immunization serum, and different H3M1-5@mRNA-M1R dose groups could significantly activate the production of neutralizing antibodies. The H3M1-5@20 μg M1R mRNA activated the production of neutralizing antibodies with the highest titer, NT 50 reached 10 2.26 , which was more than 8 times that of the negative control group (see Figure 22 ).

[0103] ELISpot assay of H3M1-5@20 μg mRNA-M1R induced cellular immune response. The splenocytes of immunized mice were stimulated with M1R full-length overlapping peptide library (synthesized from Shengong Bioengineering), and the number of interferon-γ (IFN-γ) positive cells was quantitatively detected after 48 hours of culture. The results showed that H3M1-5@mRNA-M1R vaccine significantly activated cellular immunity, inducing about 240 spots per million splenocytes, which was more than 5 times that of the negative control group (see Figure 23 ).

[0104] (3) Application of H3M1-5 polypeptide carrier in tumor mRNA vaccine

[0105] A B16-OVA tumor model was constructed based on a chicken ovalbumin (OVA)-overexpressing melanoma B16 cell line, and mRNA-OVA encoding OVA antigen was synthesized to evaluate the anti-tumor effect of mRNA vaccine. H3M1-5 and mRNA-OVA were mixed at a N / P ratio of 2:1 in 20 mM HEPES (pH 7.4) buffer to form H3M1-5@mRNA-M1R complexes. Six to eight-week-old female C57BL / 6 mice were intradermally inoculated with three doses of vaccine at 0, 7, and 14 days, respectively, 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, wherein LNP@5 μg mRNA-OVA was a liposome nanoparticle positive control group (LNP preparation method referred to CN117821482A), and PBS was a negative control group. Seven days after the last immunization, the mouse serum was collected to detect OVA-specific antibody titers, and the mouse spleen and lymph nodes were collected to evaluate the level of cellular immunity.

[0106] 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 , 10 3.37 , which were slightly lower than the antibody titer of LNP@5 μg OVA mRNA (10 4.24 ). ELISpot results showed (see Figure 25 ), after in vitro stimulation of splenocytes with short peptide SIINFEKL, H3M1-5@mRNA-OVA significantly activated cellular immunity, although lower than the LNP positive control, H3M1-5@10 μg mRNA-OVA still induced a high level of cellular immunity, producing about 650 IFN-γ positive spots (per million splenocytes), which was more than 30 times that of the negative control group.

[0107] In the B16-OVA melanoma treatment model, 6-8 week old female C57BL / 6 mice were subcutaneously inoculated with 3x10 5 B16-OVA melanoma cells, and 7 days later, 20 mM HEPES, LNP@5 μg mRNA-OVA, H3M1-5@5 μg mRNA-OVA, and H3M1-5@10 μg mRNA-OVA were injected intradermally, respectively. The immunization process was intradermal inoculation for three doses at 0, 7, and 14 days. The change in tumor volume of the mice was measured by 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 tumor inhibition effect compared with the LNP positive control group. The negative control group and the experimental group were dissected on the 20th day and the 23rd day, respectively, and the tumor weight was measured. The tumor weight and size 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 ).

[0108] (4) Element splicing enhances the immunogenicity of polypeptide biomimetic carrier mRNA vaccine

[0109] The polypeptide carrier has the advantage of modular splicing of delivery elements, which can further enhance the delivery efficiency of the candidate polypeptide. The 22A peptide (sequence PVLDLFRELLNELLEALKQKLK (SEQ ID NO. 22)) is a lipoprotein-mimicking polypeptide composed of 22 amino acids, which has certain lymph node targeting and adjuvant effect. The 22A peptide is fused to the N-terminus of H3M1-5, and the 22A-H3M1-5 fusion peptide is chemically synthesized (synthesized by Shengong Bio). In 20 mM HEPES (pH 7.4) buffer, 22A-H3M1-5 and mRNA-OVA are mixed at an N / P ratio of 2:1 to form 22A-H3M1-5@mRNA-M1R complexes.

[0110] In BALB / c mice, 20 mM HEPES, H3M1-5@20 μg mRNA-M1R, and 22A-H3M1-5@20 μg mRNA-M1R were intradermally inoculated at 0, 7, and 14 days. Seven days after the last immunization, ELISA results showed that the 22A-H3M1-5 immunization group (average titer 10 5.61 ) showed a stronger antibody response than the H3M1-5 group (average titer 10 4.85 ), with M1R-specific IgG antibody titers increased by more than 5 times (see Figure 27 ).

[0111] The immunoprotective efficacy of candidate monkeypox mRNA vaccines was evaluated in a mousepox-induced lethal challenge model (refer to CN117821482A). Fourteen days after the last immunization, mice were intraperitoneally injected with 50 PFU of mousepox virus. Compared to the control group, which all died within 6 days, mice immunized with H3M1-5@20 μg mRNA-M1R and 22A-H3M1-5@20 μg mRNA-M1R achieved survival rates of 80% and 100%, respectively, indicating that both peptide-based vector vaccines possess high immunoprotective efficacy (see [link to relevant documentation]). Figure 28 ).

[0112] Example 5. Safety evaluation of biomimetic polypeptide vector mRNA vaccine

[0113] Previous studies have shown that LNP has high pro-inflammatory activity, which adversely affects the safety of LNP-mRNA vaccines. In a BALB / c mouse model, the safety of LNP and peptide-based mRNA was compared. Single-dose intradermal administration of naked mRNA-OVA was performed on the control group, LNP@5 μg mRNA-OVA, H3M1-5@10 μg mRNA-OVA, and 22A-H3M1-5@10 μg mRNA-OVA. Serum samples were collected 4 hours later, and the levels of inflammatory cytokines were measured. 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 in the control group; for example, the level of IL-6 was more than 10 times that of the control group. However, there was no significant difference between the H3M1-5 and 22A-H3M1-5 immunized groups and the control group, and no significant inflammatory cytokines were activated (see [link to relevant documentation]). Figure 29 ).

[0114] Furthermore, LNP delivery in vivo exhibits hepatotropism, readily accumulating in the liver and posing a potential safety risk. Single-needle intradermal immunization with LNP@1μg mRNA-Fluc and 22A-H3M1-5@5μg mRNA-Fluc, followed by in vivo or organ imaging (heart, liver, spleen, lung, and kidney) in mice 24 hours later, showed that LNP@mRNA-Fluc, in addition to signal enhancement at the injection site, also significantly accumulated in the liver; while 22A-H3M1-5@mRNA-Fluc only accumulated at the injection site and did not migrate to other organs (see [link to study]). Figure 30 ).

[0115] Blood biochemical parameters were then measured after multiple immunizations. BALB / c mice were intradermally inoculated on days 0, 7, and 14. 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 analysis. The results showed no difference in ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), and URA (urea) levels between the immunization groups and the control group (see [reference]). Figure 31 ).

Claims

1. A polypeptide variant based on the ARC protein, characterized in that, The aforementioned ARC protein-based polypeptide variant is a variant based on spatial conformation design, positively charged amino acid point mutations, and side chain modification with a lipid group or a lipid derivative. The spatial conformation is an α-helix, the positively charged amino acid point mutations aim to raise the isoelectric point to above 10.0, and the lipid group or lipid derivative is octadecyl. The structure of the ARC protein-based polypeptide variant is as follows: PLRQFLWRKRRLYQTLY-GSG-K (C18).

2. The polypeptide variant based on the ARC protein according to claim 1, characterized in that, The N-terminus of the ARC protein-based polypeptide variant is also directly fused with a polypeptide having a targeting function and / or adjuvant effect, the sequence of which is shown in SEQ ID NO.

22.

3. The use of the polypeptide variant based on the ARC protein as described in claim 1 or 2 in the preparation of gene editing vectors or mRNA vaccines.

Images