A polypeptide and its polypeptide complex nanoparticle, nucleic acid vaccine and application thereof

Abstract

The present invention provides a polypeptide and polypeptide complex nanoparticles, a nucleic acid vaccine and applications thereof, belonging to the field of drug delivery. The polypeptide has the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acids to penetrate cell membranes, and having a higher antibody effect. The polypeptide and polypeptide complex nanoparticles containing the polypeptide can be used for in vitro and in vivo cell gene transfection and for the preparation of vaccine preparations.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery, and in particular to a polypeptide and polypeptide complex nanoparticles, a nucleic acid vaccine and applications thereof. Background Art

[0002] Gene transfection is a technology that transfers or delivers nucleic acids with biological functions into cells and allows the nucleic acids to maintain their biological functions in cells. Gene vectors refer to a tool for introducing exogenous therapeutic genes into biological cells. Nucleic acid vaccines are a new type of vaccine developed in recent years. As a new response plan, nucleic acid vaccines introduce nucleic acids encoding antigenic proteins into cells, synthesize proteins through the cell's expression system, and thus induce specific immune responses. Although most cells can spontaneously take up nucleic acids, the efficiency is very low and saturates at low doses. In addition, due to the presence of a large number of RNases (RNases) in nature, RNA is very unstable both in vivo and in vitro and is easily degraded. Therefore, suitable preparations are needed to protect nucleic acids from extracellular RNase-mediated degradation and promote their entry into cells. In the development of preventive and therapeutic nucleic acid vaccines, it is crucial to deliver nucleic acids of specific sequences to dendritic cells (DCs) to enable their safe, efficient, and sufficient expression for the efficacy of vaccines.

[0003] Cell penetrating peptides (CPPs) are a class of polypeptides that can directly penetrate the cell membrane into the cell in a non-receptor-dependent and non-classical endocytic manner without causing cell membrane damage. They are generally less than 30 amino acids in length and rich in basic amino acids. The amino acid sequence is usually positively charged, such as human immunodeficiency virus-1 transcription activator TAT (human immunodeficiency virus-1 transcription activator, HIV-1 TAT) (Vives et al., J. Biol. Chem. 1997; 272, 16010). The common properties of CPPs are: net positive charge or neutral charge, hydrophilicity and hydrophobicity (amphiphilicity); high transmembrane delivery efficiency; low cytotoxicity; no cell type restrictions; different bioactive substances can be introduced into cells by chemical binding or gene fusion, making it a potential multifunctional targeted drug carrier. Different CPPs have different specific transmembrane mechanisms. Studies have found that specific amino acid sequences can bind to mRNA and interfere with and reduce cell membrane stability, thereby carrying bioactive substances through the cell membrane, such as the arginine-alanine-leucine-alanine residue (RALA) sequence (Pardi et al., Curr Opin in Immunol. 2020, 65: 14-20). Compared with other delivery methods, there is still a lack of reports on the research of CPPs in the field of mRNA delivery or vaccine development.

[0004] The advantage of preparing polypeptide complex nanoparticles by connecting polypeptides via amide bonds (peptide bonds) is that they can ensure high transfection efficiency and low cytotoxicity while being able to be degraded into amino acids in vivo. However, there is currently no polypeptide complex nanoparticle delivery system for mRNA gene drugs to be marketed.

[0005] In view of the shortcomings of existing delivery systems, the present invention synthesizes non-naturally occurring polypeptides and prepares polypeptide complex nanoparticles to provide improved gene vectors for mRNA delivery and nano-delivery solutions suitable for the development of animal vaccines or human mRNA drugs. Summary of the invention

[0006] Brief description of the invention

[0007] To solve the above problems, in the first aspect, the present invention provides a polypeptide compound for nucleic acid drug delivery. In the second aspect, the present invention provides a polypeptide complex nanoparticle comprising the polypeptide compound. In the third aspect, the present invention provides an application of a polypeptide complex nanoparticle in in vivo and in vitro nucleic acid delivery. In the fourth aspect, the present invention provides a nucleic acid vaccine containing the polypeptide complex nanoparticle. In the fifth aspect, the present invention provides a use of the polypeptide complex nanoparticle in the preparation of a drug or a kit. DETAILED DESCRIPTION OF THE INVENTION

[0008] In a first aspect, the present invention provides a polypeptide compound having the following general structure:

[0009] (Xaa) x -Arg-Val-Gln-Pro-Thr-Glu-Ser-Ile-Val-Arg-(Yaa) y (Formula I),

[0010] Wherein: x is an integer from 1 to 25, and y is an integer from 0 to 9;

[0011] (Xaa) x It can be a polypeptide segment composed of any amino acids.

[0012] In some embodiments, Xaa is selected from at least one of Arg (R), Trp (W), Cys (C), Lys (K), Leu (L), Phe (F), Pro (P) or His (H), and x is the number of amino acids, and x is an integer from 1 to 20. In some embodiments, (Xaa) x In some embodiments, (Xaa) x is (Xa'a') n (Arg) 1-10 (Xa'a') n, wherein Xa'a' is selected from at least one of Arg (R), Trp (W), Cys (C), Lys (K), Leu (L), Phe (F), Pro (P) or His (H), and n is an integer from 0 to 10. In certain embodiments, Xaa is (Arg) 1-10 , Trp (W) and / or Cys (C). In other embodiments, Xaa consists of (Arg) 1-10 , Trp(W), Cys(C), His(H), and / or Pro(P), Trp(W), Cys(C), His(H), and / or Pro(P) can be in the (Arg) 1-10 It can also be preceded by (Arg) 1-10 After, or interspersed with one or more (Arg) 1-10 middle.

[0013] In the general formula I, the order of Arg (R), Trp (W), Cys (C), Lys (K), Leu (L), Phe (F), Pro (P) or His (H) in the amino acid sequence is not limited.

[0014] (Yaa)y is a polypeptide segment composed of any amino acids; in some embodiments, Yaa is selected from at least one of Arg (R), Trp (W), Phe (F) or Cys (C), y is the number of amino acids, and y is an integer between 0 and 10.

[0015] The x can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25.

[0016] The y can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0017] The n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0018] In some embodiments, the amino acid sequence of the polypeptide compound is: Seq.01, Seq.02, Seq.03, Seq.04, Seq.05, Seq.06, Seq.07, Seq.08, Seq.09, Seq.10, Seq.11, Seq.12, Seq.13, Seq.14, Seq.15, Seq.16, Seq.17, Seq.18, Seq.19, Seq.20, Seq.21, Seq.22, Seq.23, Seq.24, Seq.25 , Seq.26, Seq.27, Seq.28, Seq.29, Seq.30, Seq.31, Seq.32, Seq.33, Seq.34, Seq.35, Seq.36, Seq.37, Seq.38, Seq.39, Seq.40, Seq.41, Seq.42, Seq.43, Seq.44, Seq.45, Seq.46, Seq.47, Seq.48, Seq.49, Seq.50, Seq.51, Seq.52 or Seq.53. In some preferred embodiments, the amino acid sequence of the polypeptide compound is: Seq.05, Seq.12, Seq.46, Seq.47, Seq.49 or Seq.53.

[0019] In some embodiments, Formula (I) is at least 50% similar to any of Seq. 01-Seq. 53 and improves delivery of a nucleic acid molecule into a cell by at least 20%.

[0020] In some embodiments, Formula (I) is at least 75% similar to any one of Seq. 01-Seq. 53 and improves delivery of a nucleic acid molecule into a cell by at least 50%.

[0021] In some embodiments, Formula (I) is at least 90% similar to any one of Seq. 01-Seq. 53 and improves delivery of a nucleic acid molecule into a cell by at least 100%.

[0022] In some embodiments, Formula (I) is at least 90% similar to any of RRRRRWCRVQPTESIVR, RRRRRWFCRVQPTESIVR, FCRWCRRVQPTESIVRRCWRCF, FCRWCRRVQPTESIVCWRRRCF, HKRWCRRWCRVQPTESIVRC, or WCRRRVQPTESIVRRRWC.

[0023] In some embodiments, the polypeptide represented by the general formula (I) comprises 10-35 amino acids, which is characterized in that it improves the delivery of nucleic acid molecules into cells by at least 10%. In some embodiments, the polypeptide represented by the general formula (I) comprises 10-35 amino acids, which is characterized in that it improves the delivery of nucleic acid molecules into cells by about 50% to about 100%. In some embodiments, the polypeptide represented by the general formula (I) comprises 10-35 amino acids, which is characterized in that it improves the delivery of nucleic acid molecules into cells by about 75% to about 500%.

[0024] The present invention provides novel non-naturally occurring polypeptides, which have the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acids to penetrate cell membranes, etc., as well as polypeptide complex nanoparticles containing the polypeptides, methods of using the same for in vivo and in vitro cell gene transfection, and methods of applying the same to vaccine preparations.

[0025] In a second aspect, the present invention provides a polypeptide complex nanoparticle.

[0026] A polypeptide complex nanoparticle, comprising:

[0027] a) at least one polypeptide according to the first aspect of the present invention, and

[0028] b) Nucleic acids.

[0029] In some embodiments, a polypeptide complex nanoparticle comprises:

[0030] a) a combination of at least one polypeptide according to the first aspect of the present invention and at least one auxiliary material; and

[0031] b) Nucleic acids.

[0032] The nucleic acid can be a chemically modified or unmodified DNA, a single-stranded or double-stranded DNA, a coding or non-coding DNA. In some embodiments, the nucleic acid is selected from a plasmid, an oligodeoxynucleotide, a genomic DNA, a DNA primer, a DNA probe, an immunostimulatory DNA, an aptamer, or any combination thereof.

[0033] The nucleic acid can be chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA. In some embodiments, the nucleic acid is selected from messenger RNA (mRNA), oligoribonucleotides, viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immunostimulatory RNA (isRNA), microRNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA bait, antisense RNA, ribozyme, or any combination thereof. In some preferred embodiments, the nucleic acid is chemically modified messenger RNA (mRNA).

[0034] The nucleic acid sequence of the RNA may include all nucleic acid sequences listed in patent US9254311B2, and also include all sequences listed in the long sequence appendix of the patent. In some embodiments, the RNA sequence of the present invention can be obtained by the nucleic acid synthesis method listed in patent US9254311B2 or CN106659803A.

[0035] The polypeptide complex nanoparticles can encapsulate mRNA and allow it to be effectively introduced into different cell lines in vitro, and can be effectively transfected in vivo. The polypeptide complex nanoparticles of the present invention can carry mRNA encoding immunogenic peptides into cells, and effectively release mRNA, express antigens and effectively achieve the purpose of immunotherapy or immunoprevention.

[0036] The present invention provides novel non-naturally occurring polypeptides, which have the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acids to penetrate cell membranes, etc., as well as polypeptide complex nanoparticles containing the polypeptides, methods of using the same for in vivo and in vitro cell gene transfection, and methods of applying the same to vaccine preparations.

[0037] In some embodiments of the present invention, a polypeptide complex nanoparticle comprises:

[0038] a) at least one polypeptide according to the present invention,

[0039] b) nucleic acids, and

[0040] c) Supplementary materials.

[0041] The auxiliary materials may include lipids or PEG derivatives.

[0042] The lipid may be a naturally occurring or artificially synthesized phospholipid or a structured lipid.

[0043] The PEG derivative can be a poloxamine, a poloxamine derivative, a poloxamer, a poloxamer derivative or a PEG lipid.

[0044] The poloxamine of the present invention can be selected from at least one of the following poloxamines: 304, 701, 704, 707, 803, 901, 904, 908, 1107, 1301, 1304, 1307, 90R4 or 150R1; the poloxamine derivative is synthesized with reference to patent CN111285845B.

[0045] The poloxamer of the present invention can be selected from at least one of the following poloxamers: poloxamer 101, poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184, poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer Poloxamer 217, Poloxamer 231, Poloxamer 234, Poloxamer 235, Poloxamer 237, Poloxamer 238, Poloxamer 282, Poloxamer 284, Poloxamer 288, Poloxamer 331, Poloxamer 333, Poloxamer 334, Poloxamer 335, Poloxamer 338, Poloxamer 401, Poloxamer 402, Poloxamer 403, and Poloxamer 407.

[0046] The phospholipids may be selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DL ... Phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 LysoPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonic acid-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-racem-(1-glycerol) sodium salt (DOPG), sphingomyelin, or phosphatidylcholine (PC) and mixtures thereof;

[0047] The structural lipid may be selected from the group consisting of cholesterol (Chol), coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof;

[0048] The PEG lipid can be selected from any one of the PEG lipids described in Patent Nos. CN111281981B, CN111315359A, and CN111356444A, for example: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-modified cholesterol, such as 1,2-dimyristoyl-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG), such as mPEG5000-C-CLS (PEG-CLS), such as mPEG2000-DSPE (PEG-DSPE).

[0049] The structural formula of the 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) is shown below:

[0050]

[0051] The structural formula of the mPEG5000-C-CLS (PEG-CLS) is shown below:

[0052]

[0053] The structural formula of the mPEG2000-DSPE (PEG-DSPE) is shown below:

[0054]

[0055] The mass ratio of the nucleic acid to the polypeptide may be less than or equal to about 1:1. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:1-about 1:52. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:48. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:40. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:32. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:24. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:16. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:10. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:8. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:5. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:4. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is about 1:1, 1:2, 1:3, 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, or 1:52.

[0056] In some embodiments, the amino acid sequence of the polypeptide is Seq.05, and the mass ratio of the nucleic acid to the polypeptide is less than or equal to 1:4. In some embodiments, the amino acid sequence of the polypeptide is Seq.05, and the mass ratio of the nucleic acid to the polypeptide is about 1:4-about 1:52.

[0057] In some embodiments, the amino acid sequence of the polypeptide is Seq. 12, and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1: about 4. In some embodiments, the amino acid sequence of the polypeptide is Seq. 12, and the mass ratio of the nucleic acid to the polypeptide is about 1: 4-about 1: 52.

[0058] In some embodiments, the amino acid sequence of the polypeptide is Seq.46, and the mass ratio of the nucleic acid to the polypeptide is less than or equal to 1:2. In some embodiments, the amino acid sequence of the polypeptide is Seq.46, and the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:52.

[0059] In some embodiments, the amino acid sequence of the polypeptide is Seq.47, and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1:2. In some embodiments, the amino acid sequence of the polypeptide is Seq.47, and the mass ratio of the nucleic acid to the polypeptide is about 1:2-about 1:52.

[0060] In some embodiments, the amino acid sequence of the polypeptide is Seq.49, and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1: 16. In some embodiments, the amino acid sequence of the polypeptide is Seq.49, and the mass ratio of the nucleic acid to the polypeptide is about 1: 16-about 1: 52.

[0061] In some embodiments, the amino acid sequence of the polypeptide is Seq.53, and the mass ratio of the nucleic acid to the polypeptide is less than or equal to about 1:4. In some embodiments, the amino acid sequence of the polypeptide is Seq.53, and the mass ratio of the nucleic acid to the polypeptide is about 1:4-about 1:52.

[0062] The mass ratio of the nucleic acid to the auxiliary material may be less than or equal to 1:2. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material may be less than or equal to about 1:50. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2-about 1:800. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2-about 1:500. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2-1:400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2-about 1:50. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1:2-about 1:33. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2-1:10. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2-1:6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2, 1:3, 1:4, 1:5 or 1:6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:33-1:400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:50-1:800.

[0063] In some embodiments, the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is 1:50-1:800. In some embodiments, the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is 1:100, 1:200 or 1:500. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is 1:2, the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is 1:50-1:800.

[0064] In some embodiments, the auxiliary materials are PEG derivatives, phospholipids and structural lipids, and the mass ratio of the nucleic acid to the auxiliary materials is about 1:2-about 1:6. In some embodiments, the auxiliary materials are PEG derivatives, phospholipids and structural lipids, and the mass ratio of the nucleic acid to the auxiliary materials is about 1:2, 1:3, 1:4, 1:5 or 1:6. In some embodiments, the mass ratio of the nucleic acid to the polypeptide is 1:2-1:30, and the auxiliary materials are PEG derivatives, phospholipids and structural lipids, and the mass ratio of the nucleic acid to the auxiliary materials is 1:2-1:6.

[0065] In some embodiments, the auxiliary material is a PEG derivative and a phospholipid, and the mass ratio of the nucleic acid to the auxiliary material is about 1:33-about 1:400 or about 1:33-about 1:370. In some embodiments, the auxiliary material is a PEG derivative and a phospholipid, the mass ratio of the nucleic acid to the auxiliary material is about 1:33-about 1:400 or about 1:33-about 1:370, and the mass ratio of the PEG derivative to the phospholipid is 32:1-700:1. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is 1:2, the auxiliary material is a PEG derivative and a phospholipid, the mass ratio of the nucleic acid to the auxiliary material is 1:33-1:400 or 1:33-1:370, and the mass ratio of the PEG derivative to the phospholipid is 32:1-700:1.

[0066] In some embodiments, a polypeptide complex nanoparticle comprises: a nucleic acid, a polypeptide compound having an amino acid sequence of Seq.05, and an auxiliary material, wherein the auxiliary material is 90R4 and lecithin; the nucleic acid and the polypeptide compound having the amino acid sequence of Seq.05, The mass ratio of 90R4 and lecithin is approximately 1:2:322:1.

[0067] In some embodiments, a polypeptide complex nanoparticle comprises: a nucleic acid, a polypeptide compound having an amino acid sequence of Seq.49, and auxiliary materials, wherein the auxiliary materials are 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol; the mass ratio of the nucleic acid, the polypeptide compound having an amino acid sequence of Seq.49, 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol is approximately 10:300:8:16:31.

[0068] In some embodiments, a polypeptide complex nanoparticle comprises: a nucleic acid, a polypeptide compound having an amino acid sequence of Seq.53, and auxiliary materials, wherein the auxiliary materials are 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol; the mass ratio of the nucleic acid, the polypeptide compound having an amino acid sequence of Seq.53, 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol is approximately 10:40:8:16:5.

[0069] In some embodiments of the present invention, a polypeptide complex nanoparticle comprises at least one non-naturally occurring peptide and a nucleic acid as described in the present invention.

[0070] In some embodiments of the present invention, a polypeptide complex nanoparticle comprises at least one non-naturally occurring peptide, a nucleic acid and at least one lipid or PEG derivative as described in the present invention.

[0071] The nucleic acid can be a chemically modified or unmodified DNA, a single-stranded or double-stranded DNA, a coding or non-coding DNA. In some embodiments, the nucleic acid is selected from a plasmid, an oligodeoxynucleotide, a genomic DNA, a DNA primer, a DNA probe, an immunostimulatory DNA, an aptamer, or any combination thereof.

[0072] The nucleic acid can be chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA. In some embodiments, the nucleic acid is selected from messenger RNA (mRNA), oligoribonucleotides, viral RNA, replicon RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), immunostimulatory RNA (isRNA), microRNA, small interfering RNA (siRNA), small nuclear RNA (snRNA), small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA bait, antisense RNA, ribozyme, or any combination thereof. In some preferred embodiments, the nucleic acid is chemically modified messenger RNA (mRNA).

[0073] The nucleic acid sequence of the RNA may include all nucleic acid sequences listed in patent US9254311B2, and also include all sequences listed in the long sequence appendix of the patent. In some embodiments, the RNA sequence of the present invention can be obtained by the nucleic acid synthesis method listed in patent US9254311B2 or CN106659803A.

[0074] In some embodiments, the polypeptide complex nanoparticles may further comprise at least one pharmaceutically acceptable excipient.

[0075] The polypeptide complex nanoparticle of the present invention is stable in aqueous solution, and can contact with the cell tissue of human or animal after formation, or can store a period of time before contacting with the cell or tissue. The polypeptide complex nanoparticle is stable, and can store and continue at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, at least 7 days, at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months or at least 1 year time period. It should be understood that the storage period can be between any one of these time periods, for example between 31 minutes and 1 hour or between 1 hour and 24 hours.

[0076] In a third aspect, the present invention provides a use of polypeptide complex nanoparticles in in vivo and in vitro nucleic acid delivery.

[0077] A use of the polypeptide complex nanoparticles according to the second aspect in in vivo and in vitro nucleic acid delivery.

[0078] The polypeptide complex nanoparticles can encapsulate mRNA and allow it to be effectively introduced into different cell lines in vitro, and can be effectively transfected in vivo. The polypeptide complex nanoparticles of the present invention can carry mRNA encoding immunogenic peptides into cells, and effectively release mRNA, express antigens and effectively achieve the purpose of immunotherapy or immunoprevention.

[0079] The present invention provides novel non-naturally occurring polypeptides, which have the functions of compressing and protecting nucleic acids from degradation, promoting nucleic acids to penetrate cell membranes, etc., as well as polypeptide complex nanoparticles containing the polypeptides, methods of using the same for in vivo and in vitro cell gene transfection, and methods of applying the same to vaccine preparations.

[0080] In a fourth aspect, the present invention provides a nucleic acid vaccine comprising the polypeptide complex nanoparticles described in the second aspect.

[0081] A nucleic acid vaccine, comprising the polypeptide complex nanoparticles described in the second aspect.

[0082] The polypeptide complex nanoparticle may include at least one RNA.

[0083] The nucleic acid vaccine can be used to treat or prevent diseases.

[0084] The RNA at least includes coding RNA.

[0085] The encoding RNA may include RNA that encodes at least one coding region of at least one therapeutic protein, therapeutic polypeptide, immunogenic protein, or immunogenic peptide. In some embodiments, the encoding RNA is mRNA.

[0086] The present invention provides a ribonucleic acid vaccine with RNA (such as messenger RNA (mRNA)) as the core and polypeptide complex nanoparticles as the delivery carrier, which can safely induce the body's naturally existing specific immune system to produce almost any target protein or its fragment, including vaccines for infectious pathogens such as bacteria and viruses and tumor vaccines. In some embodiments, the RNA is modified. The nucleic acid vaccine disclosed in the present invention can be used to induce immune responses to infectious pathogens or cancers, including cellular immune responses and humoral immune responses, without the risk of insertional mutagenesis. Depending on the incidence of infectious pathogens and cancers, nucleic acid vaccines with polypeptide complex nanoparticles as delivery carriers can be used in various types of diseases. The nucleic acid vaccine can be used to prevent and / or treat infectious pathogens or cancers of various metastatic stages or degrees.

[0087] In some embodiments of the present invention, a nucleic acid vaccine comprises the polypeptide complex nanoparticles described in the second aspect; the polypeptide complex nanoparticles include at least one RNA; the RNA is messenger RNA (mRNA); the messenger RNA (mRNA) can safely guide the body's cellular machinery to produce almost any target protein, from natural proteins to antibodies and other completely novel protein constructs that may have therapeutic activity inside and outside cells.

[0088] The nucleic acid vaccine can be used in various situations according to the prevalence of infection or the degree or level of unmet medical needs. The nucleic acid vaccine can be used to treat and / or prevent HPV of various genotypes, strains and isolates. The superiority of the nucleic acid vaccine is that it produces much greater antibody titers than commercially available antiviral treatments and produces reactions earlier. Although it is not desired to be bound by theory, it is believed that like mRNA polynucleotides, RNA vaccines are better designed to produce appropriate protein configurations by translation when RNA vaccines assign natural cell mechanisms. Unlike traditional vaccines that are manufactured in vitro and can trigger adverse cellular responses, the nucleic acid vaccine provides a template for expressing protein antigens in a more natural way to the cell system.

[0089] In some embodiments of the present invention, a nucleic acid vaccine comprises the polypeptide complex nanoparticles of the second aspect; the polypeptide complex nanoparticles include at least one RNA; the nucleotide sequence of the RNA is a nucleotide sequence encoding an antigen of any pathogen. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is mRNA, and the nucleotide sequence of the mRNA encodes the S spike protein of the new coronavirus SARS-CoV-2.

[0090] In some embodiments of the present invention, a nucleic acid vaccine comprises the polypeptide complex nanoparticles of the second aspect; the polypeptide complex nanoparticles contain artificially synthesized pathogen antigen polypeptides. In some embodiments, the antigen polypeptides are fused with other polypeptides that can enhance transfection and delivery efficiency and / or enhance immune response.

[0091] The dosage form of the nucleic acid vaccine can be injection, tablet, inhalation preparation, suppository, eye drops or suspension, etc.

[0092] The nucleic acid vaccine of the present invention can be administered by any route that produces a therapeutically effective result. The route includes (but is not limited to) intradermal, subcutaneous, intraperitoneal, oral, intramuscular, intranasal, intraocular, upper respiratory tract, intravenous, vaginal, and rectal administration. In some embodiments, the mRNA vaccine of the present invention is administered by injection.

[0093] In a fifth aspect, the present invention provides a use of the polypeptide complex nanoparticles described in the second aspect in the preparation of a drug or a kit.

[0094] A use of the polypeptide complex nanoparticles according to the second aspect in the preparation of a drug or a kit.

[0095] In some embodiments, a polypeptide complex nanoparticle as described in the second aspect is used in the preparation of a medicament for preventing, treating and / or ameliorating a disease selected from the following: cancer or tumor disease, infectious disease, autoimmune disease, allergy or allergic disease, single gene genetic disease, or general genetic disease, disease with a genetic background and typically caused by a determined gene defect and inherited according to Mendel's law, cardiovascular disease, neuronal disease, respiratory disease, digestive system disease, skin disease, musculoskeletal disorder, connective tissue disorder, tumor, immunodeficiency, endocrine, nutritional and metabolic diseases, eye disease and ear disease.

[0096] The infectious disease may include a viral infectious disease, a bacterial infectious disease or a protozoological infectious disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1The transmission electron microscopy image of the polypeptide complex nanoparticles described in Example 3 is shown; in the image, A represents the prescription Rp.05, B represents the prescription Rp.28, C represents the prescription Rp.43, and the white scale is 200nm.

[0098] Figure 2 The agarose gel electrophoresis results of the polypeptide complex nanoparticles in Example 4 are shown; in the figure, mRNA refers to the mRNA positive control group, 1, 2, 4, 8, 16, 32, and 64 refer to - the mass ratios of polypeptide to mRNA are 1:1, 2:1, 4:1, 8:1, 16:1, 32:1, and 64:1; the minimum mass ratios of each polypeptide to ensure complete compression of mRNA are: Seq.05 is 4, Seq.12 is 4, Seq.46 is 2, Seq.47 is 2, Seq.49 is 16, and Seq.53 is 4.

[0099] Figure 3 The figure shows the transfection of the FLuc-mRNA polypeptide complex nanoparticles in DC2.4 cells in Example 5; the abscissa in the figure represents polypeptide nanoparticle compositions of different prescriptions, and the ordinate represents the relative fluorescence intensity expressed 24 hours after transfection of the polypeptide nanoparticle composition containing the same dose of FLuc-mRNA.

[0100] Figure 4 The figure shows the survival rate of DC2.4 cells after being treated with different prescriptions in Example 5; the horizontal axis represents different prescriptions of polypeptide complex nanoparticles, and the vertical axis represents cell viability. The higher the cell activity, the lower the cytotoxicity.

[0101] Figure 5 The figure shows the transfection of Luc-pDNA polypeptide complex nanoparticles in DC2.4 cells in Example 5; the horizontal axis represents different prescriptions, and the vertical axis represents the relative fluorescence intensity expressed by DC2.4 cells 24h, 48h, and 72h after transfection with the same dose of Luc-pDNA.

[0102] Figure 6 Example 6 shows the IVIS detection of the expression of luciferase in mice using polypeptide complex nanoparticles.

[0103] Figure 7 The serum IgG antibody levels of mice after immunization with the polypeptide complex nanoparticles in Example 7 are shown; the horizontal axis represents the 28th and 49th days after the first immunization of different prescriptions, and the vertical axis represents the difference in OD values ​​of optical density at two wavelengths. The OD value is an indicator for judging the IgG antibody level in serum, reflecting the level of anti-S protein IgG in serum.

[0104] Figure 8The serum IgG antibody titer of mice immunized with polypeptide complex nanoparticles in Example 7 is shown; the horizontal axis represents the different dilution multiples of the serum 49 days after the first immunization of different prescriptions, and the vertical axis represents the difference in OD values ​​(optical density values) at two wavelengths. 2x Baseline (twice the background) is used as the dividing value for distinguishing positive and negative results, and the maximum dilution multiple with an OD value higher than this value is the titer.

[0105] Definition of terms

[0106] The terms used throughout this specification generally have their general meaning in the art in the context of the present invention and in the specific circumstances in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide practitioners with other guidance on describing various embodiments of the present invention and how to form and use the embodiments. It should be understood that the same concept can be expressed in more than one way. Therefore, alternative language and synonyms can be used for any one or more of the terms discussed herein, regardless of whether a term is described or discussed herein, and no specific meaning is given. Synonyms for certain terms can be provided. Narrating one or more synonyms does not exclude the use of other synonyms. The use of examples (including examples of any term discussed herein) anywhere in this specification is only illustrative, and in no way limits the scope and meaning of the present invention or any exemplified term.

[0107] In the above text of the present invention, whether or not the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. The value of each number may have a difference of less than 10% or a reasonable difference considered by those skilled in the art, such as a difference of 1%, 2%, 3%, 4% or 5%.

[0108] The term "polypeptide" It refers to a polymer of amino acid residues (natural or non-natural) that are often linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides can be unimolecular or can be multimolecular complexes, such as dimers, trimers or tetramers. They can also contain single-chain or multi-chain polypeptides such as antibodies or insulin, and can be associated or linked. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0109] The term "protein"It is a polymer consisting essentially of any of the 20 amino acids. Although "polypeptide" is often used to refer to relatively large polypeptides, and "peptide" is often used to refer to small polypeptides, the use of these terms in the art overlaps and varies. The terms "peptide," "protein," and "polypeptide" are sometimes used interchangeably herein.

[0110] The term "hydrophilic" It means that it can be dissolved in water under specific conditions, including easily soluble in water, soluble in water, and slightly soluble in water.

[0111] The term "hydrophobic" It means that it is difficult to dissolve in water under certain conditions.

[0112] As used herein, the term "amino acid" generally refers to naturally occurring or synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure (i.e., an α carbon bound to a hydrogen, carboxyl, amino group, and R group) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine, and methylsulfonium. Such analogs have modified R groups (e.g., norleucine or norvaline) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds having a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to naturally occurring amino acids. The term "amino acid" may refer to an amino acid or a derivative thereof (e.g., an amino acid analog), as well as its D and L forms. Examples of such amino acids include glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, N-acetylcysteine.

[0113] "Kit" refers to a transfection, DNA, RNAi or other bioactive (e.g., protein or anionic molecule) delivery or protein expression or gene knockdown kit, which includes one or more reagents of the present invention or a mixture thereof. The kit may include one or more non-naturally occurring peptides described herein, or optionally a composition with one or more lipids or PEG derivatives. In some embodiments, peptides and lipid reagents can be provided in a single formulation. In other embodiments, the composite material and peptide can be provided separately, together with instructions for instructing the user to combine the reagents when used. Such kits may include a carrying device that is separated and holds one or more container devices (such as vials, test tubes, etc.) in a tightly constrained manner. Each of such container devices contains components or component mixtures required for transfection. Such kits may optionally include one or more components selected from any bioactive molecule, such as nucleic acids (one or more expression vectors, DNA molecules, RNA molecules or RNAi molecules in some embodiments), cells, one or more compounds of the present invention, lipid compounds, transfection enhancers, bioactive substances, etc.

[0114] The culture medium, method, kit and composition of the present invention are suitable for monolayer or suspension culture, transfection and cultivation of cells, and are suitable for protein expression in monolayer or suspension cultured cells. In some embodiments, the culture medium, method, kit and composition of the present invention are used for suspension culture, transfection and cultivation of cells, and for protein product expression in suspension cultured cells.

[0115] Immune response: An immune response can typically be a specific response of the adaptive immune system to a specific antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response). (Fotin-Mleczek Mariola et al., CN108064176A)

[0116] Vaccine: A vaccine is typically understood as a prophylactic or therapeutic substance that provides at least one antigen or antigenic function. The antigen or antigenic function can stimulate the body's adaptive immune system to provide an adaptive immune response. (Fotin-Mleczek Mariola et al., CN108064176A)

[0117] mRNA providing an antigen: The mRNA providing an antigen may typically be an mRNA having at least one open reading frame, which can be translated by the cell or organism to which the mRNA is provided. The product of the translation is a peptide or protein that can be used as an antigen (preferably as an immunogen). The product may also be a fusion protein composed of more than one immunogen, such as a fusion protein composed of two or more epitopes, peptides or proteins, wherein the epitopes, peptides or proteins may be connected by a linker sequence. (Fotin-Mleczek Mariola et al., CN108064176A)

[0118] Nucleic acid: The term "nucleic acid" refers to any DNA or RNA molecule and is used synonymously with "polynucleotide". Wherever a nucleic acid or a nucleic acid sequence encoding a specific protein and / or peptide is mentioned herein, the nucleic acid or nucleic acid sequence, respectively, preferably also includes regulatory and / or other sequences that allow its expression and / or stability in a suitable host (e.g., human), i.e., transcription and / or translation of the nucleic acid sequence encoding the specific protein or peptide. (Fotin-Mleczek Mariola et al., CN108064176A)

[0119] Peptide: A peptide is a polymer of amino acid monomers. Typically, the monomers are linked by peptide bonds. The term "peptide" does not limit the length of the polymer chain of amino acids. In some embodiments of the invention, a peptide may, for example, contain less than 50 monomer units. Longer peptides, which may also be referred to as polypeptides, typically have 50 to 600 monomer units, more specifically 50 to 300 monomer units. (Fotin-Mleczek Mariola et al., CN108064176A)

[0120] Pharmaceutically effective amount: In the context of the present invention, a pharmaceutically effective amount is typically understood to be an amount sufficient to induce an immune response or trigger a desired therapeutic effect. (Fotin-Mleczek Mariola et al., CN108064176A)

[0121] Chemical synthesis of RNA: Chemical synthesis of relatively short fragments of oligonucleotides with defined chemical structures provides a fast and inexpensive method for obtaining custom oligonucleotides of any desired sequence. Although enzymes synthesize DNA and RNA only in the 5' to 3' direction, chemical oligonucleotide synthesis does not have this limitation, although it is most commonly performed in the opposite (i.e., 3' to 5') direction. Currently, the process is implemented in the form of solid phase synthesis using the phosphoramidite method and phosphoramidite building blocks derived from protected nucleotides (A, C, G and U) or chemically modified nucleotides. (Fotin-Mleczek Mariola et al., CN108064176A)

[0122] To obtain the desired oligonucleotide, the structural units are sequentially coupled to the growing oligonucleotide chain on the solid phase in the order required for the product sequence in a fully automated process. After completing the chain assembly, the product is released from the solid phase into the solution, deprotected, and collected. The presence of side reactions sets a practical limit to the length of the synthetic oligonucleotide (up to about 200 nucleotide residues) because the number of errors increases with the length of the synthesized oligonucleotide. The product is usually separated by HPLC to obtain the desired oligonucleotide of high purity. (Fotin-Mleczek Mariola et al., CN108064176A)

[0123] RNA in vitro transcription: The term "RNA in vitro transcription" or "in vitro transcription" relates to a process in which RNA is synthesized (in vitro) in a cell-free system. DNA, in particular plasmid DNA, is used as a template for the production of RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the invention is preferably a linearized plasmid DNA template. The promoter used to control the in vivo transcription can be any promoter for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3 and SP6 RNA polymerases. The DNA template for in vitro RNA transcription can be obtained by cloning a nucleic acid (in particular cDNA) corresponding to the corresponding RNA to be transcribed in vitro and introducing it into a suitable vector for in vitro transcription (e.g., into a plasmid DNA). In a preferred embodiment of the present invention, the DNA template is linearized with a suitable restriction endonuclease and subsequently transcribed in vitro. cDNA can be obtained by reverse transcription or chemical synthesis of mRNA. In addition, the DNA template for in vitro RNA synthesis can also be obtained by gene synthesis. (Fotin-Mleczek Mariola et al., CN108064176A)

[0124] Methods for in vitro transcription are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101-14).

[0125] RNA, mRNA: RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e., a polymer composed of nucleotide monomers. These nucleotides are usually adenosine monophosphate (AMP), uridine monophosphate (UMP), guanosine monophosphate (GMP) and cytidine monophosphate (CMP) monomers or their analogs, which are connected to each other along the so-called backbone. The backbone is formed by a phosphodiester bond between the sugar (i.e., ribose) of the first monomer and the phosphate portion of the second adjacent monomer. The specific order of monomers, i.e., the order of bases connected to the sugar / phosphate backbone, is called the RNA sequence. Generally, RNA can be obtained by transcription of a DNA sequence (e.g., in a cell). In eukaryotic cells, transcription is typically performed in the nucleus or mitochondria. In vivo, transcription of DNA generally produces a so-called pre-mature RNA (also called pre-mRNA, pre-mRNA or heterologous nuclear RNA), which must be processed into a so-called messenger RNA (usually abbreviated as mRNA). For example, the processing of RNA before maturity in eukaryotic organisms includes a variety of different post-transcriptional modifications, such as splicing, 5'-capping, polyadenylation, export from the nucleus or mitochondria and similar modifications. The sum of these processes is also referred to as the maturation of RNA. Mature messenger RNA generally provides a nucleotide sequence that can be translated into an amino acid sequence of a specific peptide or protein. Typically, mature mRNA comprises a 5'-cap, an optional 5'UTR, an open reading frame, optionally a 3'UTR and a poly (A) tail (Fotin-Mleczek Mariola et al., CN108064176A).

[0126] In addition to messenger RNA, there are several non-coding types of RNA that can participate in the regulation of transcription and / or translation and immunostimulation. In the present invention, the term "RNA" further includes any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecules known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA (asRNA), circular RNA (circRNA), ribozymes, aptamers, riboswitches, immunostimulatory / immunostimulatory RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA) and Piwi interacting RNA (piRNA) (Fotin-Mleczek Mariola et al., CN108064176A).

[0127] The term "chemically modified" refers to the modification of A, G, U or C ribonucleotides. Generally, in this article, these terms are not intended to refer to the modification of the ribonucleotides in the mRNA cap portion at the naturally occurring 5' end. The modification may be a variety of modifications. In some embodiments, where the nucleic acid is an mRNA, the coding region, flanking region and / or terminal region may contain one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide introduced into the cell can show reduced degradation in the cell compared to the unmodified polynucleotide.

[0128] The term "amino acid" as described herein refers to a molecule with a side chain, an amino group and an acid group (e.g., -CO2H's carboxyl group or -SO3H's sulfo group), wherein the amino acid is connected to the parent molecular group by a side chain, an amino group or an acidic group (e.g., a side chain). In some embodiments, the amino acid is connected to the parent molecular group by a carbonyl group, wherein the side chain or the amino group is connected to the carbonyl group. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclic radical, alkaryl, alkanehterocyclic radical, aminoalkyl, carbamoylalkyl and carboxylalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine and valine. The amino acid group may be optionally substituted with 1, 2, 3 or, in the case of 2 or more carbon amino acid groups, 4 substituents independently selected from: (1) C1-6 alkoxy; (2) C1-6 alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino).

[0129] Delivery: As used herein, "delivery" refers to the act or manner of delivering a compound, substance, entity, moiety, cargo, or payload.

[0130] Delivery Agent: As used herein, "delivery agent" refers to any substance that at least partially facilitates the delivery of a polynucleotide to a target cell in vivo.

[0131] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); (3) translation of the RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

[0132] Formulation: As used herein, "formulation" includes at least one polynucleotide and a delivery agent.

[0133] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reactor, in a cell culture, in a Petri dish, etc., rather than within an organism (eg, an animal, plant, or microorganism).

[0134] In vivo: The term "in vivo" as used herein refers to events that occur within an organism (e.g., an animal, plant or microorganism, or a cell or tissue thereof). Modified: "Modified" as used herein refers to an altered state or structure of a molecule of the present invention. Molecules can be modified in a variety of ways, including chemical, structural and functional modifications. In one embodiment, the mRNA molecule of the present invention is modified, for example, by the introduction of non-natural nucleosides and / or nucleotides when it relates to natural ribonucleotides A, U, G and C. Atypical nucleotides such as cap structures are not considered to be "modified", although they are different in chemical structure from A, C, G, U ribonucleotides.

[0135] Naturally occurring: As used herein, "naturally occurring" means occurring in nature without the assistance of human beings.

[0136] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0137] Preventing: As used herein, the term "preventing" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical manifestations of a specific infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or manifestations of a specific infection, disease, disorder, and / or condition; partially or completely delaying the progression of an infection, a specific disease, disorder, and / or condition; and / or reducing the risk of developing pathology associated with an infection, disease, disorder, and / or condition.

[0138] Protein of interest: As used herein, the term "protein of interest" or "desired protein" includes the proteins provided herein and fragments, mutants, variants and alterations thereof.

[0139] Treating: The term "treating" as used herein refers to partially or completely alleviating, improving, ameliorating, relieving a specific infection, disease, disorder, and / or condition; partially or completely delaying the onset of a specific infection, disease, disorder, and / or condition; partially or completely inhibiting the progression of a specific infection, disease, disorder, and / or condition; partially or completely reducing the severity of a specific infection, disease, disorder, and / or condition; and / or reducing the incidence of one or more symptoms or characteristics of a specific infection, disease, disorder, and / or condition. For example, "treating" cancer can refer to inhibiting the survival, growth, and / or spread of a tumor. For the purpose of reducing the risk of developing pathology associated with a disease, disorder, and / or condition, treatment can be applied to subjects who do not show signs of a disease, disorder, and / or condition and / or subjects who only show early signs of a disease, disorder, and / or condition. Specific embodiments

[0140] In order to enable those skilled in the art to better understand the technical solution of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0141] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0142] The amino acid sequence of the polypeptide synthesized in the embodiment of the present invention is:

[0143] Table 1: Polypeptides of the general formula of the present invention

[0144]

[0145]

[0146]

[0147] Table 1 sets forth various peptide sequences that can be used to practice the present invention, but it should be understood by those of ordinary skill in the art that the list of peptide sequences in Table 1 is provided by way of example only, and is not intended to limit the scope of the present invention to only those sequences explicitly written out. On the contrary, it will be readily apparent to such personnel that, based on the teachings set forth above regarding the polypeptides described by the general formula, there may be a large number of peptides that are potentially suitable for practicing the present invention set forth herein. In addition, it is well within the knowledge of skilled professionals to determine whether a given peptide sequence falls within the scope of the present invention using standard techniques in the art without the need for undue experimentation. In addition, it should be understood that various variants of the peptide sequences appearing in Table 1 also fall within the scope of the present invention, as long as such variants meet the structural and functional characteristics set forth above. Variants of the peptide sequences appearing in Table 1 or variants of any other candidate peptides that are not explicitly described in Table 1 but meet the structural and functional requirements set forth above may include deletions, insertions, and substitutions using naturally occurring or non-proteinogenic amino acids.

[0148] Example 1: Preparation method of the polypeptide of the present invention

[0149] The non-naturally occurring peptides of the present invention are produced by any previously known peptide synthesis method known to those of ordinary skill in the art, including but not limited to recombinant methods or peptide synthesis chemistry, such as solid phase peptide synthesis. Solid phase synthesis methods (Marrifield, Journal of the American Chemical Society (J. Am. Chem. Soc.), 85, 2149-2154, 1963) can be noted as just one example of such peptide synthesis methods. Currently, peptides can be produced simply and in a relatively short period of time using automated universal peptide synthesizers based on those principles. In addition, peptides can be produced using well-known recombinant protein production techniques, which are widely known to skilled professionals.

[0150] The simple synthesis method and specific process of the polypeptide of the present invention are described as follows (taking Seq.05 as an example):

[0151] (1) Resin treatment

[0152] ① Resin swelling: Select Fmoc-Arg(pbf)-2-Chlorotrityl Resin (molar substitution coefficient is 0.317 mmol / g) as the starting resin, add it to a 50 ml reaction column, add DCM (dichloromethane) to soak, drain and complete the swelling of the resin.

[0153] ② Deprotection: Add DMF (N,N-dimethylformamide) solution containing 20% ​​(g / 100ml) piperidine, stir for 30 minutes under N2 (nitrogen), filter and dry the solvent; then wash the resin with DMF (N,N-dimethylformamide) 6 times, drain and complete the deprotection of the resin.

[0154] (2) Amino acid coupling reaction

[0155] Reaction monitoring detection method: The reaction progress was monitored by the ninhydrin method.

[0156] The raw materials and reagents used are as follows:

[0157] name Feeding amount (mmol) Protect amino acids 2 O-Benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) 2 N,N-Diisopropylethylamine (DIEA) 2

[0158] The specific operation process is as follows:

[0159] Weigh the corresponding amount of TBTU and protected amino acid into a beaker, add DMF to dissolve; then add the reaction solution to the resin after deprotection in step (1), add DIEA, pass N2 to blow for about 90 minutes, and detect by ninhydrin reaction. After the reaction is completed, remove the solvent and wash the resin with DMF 3 times. Add a DMF solution containing 20% ​​(g / 100ml) piperidine to the resin, pass N2 to continue blowing for 30 minutes, then remove the solvent and wash the resin with DMF 6 times to complete the coupling of this amino acid.

[0160] The above reaction procedure was repeated until the condensation reaction of all protected amino acids was completed. After coupling to the last protected amino acid, the peptide was shrunk, washed with DMF, DCM, and methanol three times in sequence, and weighed after being drained.

[0161] Condensation method: TBTU+DIEA, condensation agent: TBTU: 0.64g, DIEA: 0.7ml.

[0162] Table 2: Abbreviations of amino acids and corresponding protected amino acids

[0163]

[0164]

[0165] (3) TFA cleavage (dissociate the peptide from the resin and remove the amino acid side chain protecting groups)

[0166] The resin obtained in "(2) Amino Acid Coupling Reaction" was added to the prepared lysate (containing 86% TFA, 5% EDT (ethylenedithiol), 5% thioanisole, 3% phenol and 2% pure water) and stirred for 150 minutes. The resin and the lysate were then separated, and ether was added to the separated lysate to fully precipitate the polypeptide; the crude peptide was filtered and fully washed with ether for 6 times to obtain the crude peptide, which was purified to obtain the polypeptide shown in Seq.05. Other polypeptides provided by the present invention were synthesized in this way.

[0167] (4) Simple steps for purification

[0168] The crude peptide obtained in step (3) was dissolved in 3 ml of a mixture of acetonitrile and water in a volume ratio of 1:1, clarified and filtered, and the purity and molecular weight of the peptide were detected by liquid chromatography-mass spectrometry (HPLC-MS). The results are shown in Table 1, and the purity of the peptides was above 98.00%.

[0169] Example 2: Preparation of polypeptide complex nanoparticles

[0170] The polypeptide complex nanoparticles of the present invention can be prepared by referring to the nanoparticle preparation method of patents CN111249476A, CN111281981 B, CN111281982A, CN111285845B, CN111588637A or application number 202110713076.9.

[0171] Preparation method 1: Dissolve the polypeptide in ultrapure water without nuclease to obtain a 1 mg / ml solution, then mix the polypeptide with mRNA according to the mass ratio in Table 3 and stir for 15 minutes, then let it stand to obtain polypeptide complex nanoparticles. The specific formula is shown in Table 3:

[0172] Table 3. Peptide nanoparticles formulation and their particle size, potential, and encapsulation efficiency

[0173]

[0174] Preparation method 2: According to the prescription design in Table 4, the PEG derivative auxiliary material was taken out from the -20°C refrigerator and equilibrated to 25°C, weighed at 25°C, dissolved in a nuclease-free 15mL centrifuge tube with nuclease-free ultrapure water to a concentration of 100 mg / mL, and fully oscillated on a vortex instrument for 5 minutes to obtain a stock solution A; then the lipid auxiliary material was taken out from the -20°C refrigerator and equilibrated to 25°C, weighed at 25°C, dissolved in a nuclease-free 1.5mL centrifuge tube with ethanol (chromatographic grade) at 25°C to a concentration of 10 mg / mL, and fully oscillated on a vortex instrument for 5 minutes to obtain a stock solution B.

[0175] The corresponding polypeptides in Table 4 were dissolved in nuclease-free ultrapure water to form a 5 mg / ml solution, and then mixed with mRNA in sequence according to the mass ratio shown in Table 4 for 10 minutes to obtain a stock solution C. The stock solution A and the stock solution B were mixed for 1 minute according to the mass ratio shown in Table 4, and then the stock solution C was added. After being fully oscillated with a vortex instrument for 1 minute, the polypeptide complex nanoparticles were obtained. The specific prescription is shown in Table 4:

[0176] Table 4. Peptide nanoparticles formulation and their particle size, potential, and encapsulation efficiency

[0177]

[0178] Preparation method three: weigh 5.0 mg DMG-PEG and add it into 1 mL chromatographically pure ethanol to dissolve; weigh 10.3 mg DSPC (distearoylphosphatidylcholine) and add it into 1 mL chromatographically pure ethanol to dissolve; weigh 19.4 mg cholesterol and add it into 2 mL chromatographically pure ethanol to dissolve.

[0179] The polypeptide was dissolved in nuclease-free ultrapure water to obtain a 1 mg / ml solution, and then the polypeptide and mRNA were mixed evenly in accordance with the corresponding mass ratios in Table 5. After 5 minutes, 20 mL of citrate buffer with a pH value of 5.4 was added, and the ethanol solution of DMG-PEG and DSPC was added dropwise to the citrate buffer, and then a certain volume (100 uL, 200 uL, 250 uL or 500 uL) of 19.4 mg / mL cholesterol ethanol solution was added dropwise. After stirring for 30 minutes, ethanol was removed by vacuum rotary evaporation at 40°C to obtain polypeptide complex nanoparticles. The specific prescription is shown in Table 5:

[0180] Table 5. Peptide nanoparticles formulation and their particle size, potential, and encapsulation efficiency

[0181]

[0182] Preparation method 4: Weigh 10 mg of the corresponding auxiliary material (DMG-PEG, PEG-CLS, or PEG-DSPE) in Table 6, add nuclease-free ultrapure water to dissolve it to a concentration of 1 mg / mL to obtain stock solution A. Dissolve the polypeptide in nuclease-free ultrapure water to obtain a 1 mg / ml solution to obtain stock solution B. After mixing stock solution A and stock solution B according to the corresponding mass ratio in Table 6 for 1 min, the mixed solution was mixed with mRNA for 10 min, 15 min, 30 min or 60 min, preferably 10 min, and then fully oscillated with a vortex instrument for 20 min to obtain polypeptide complex nanoparticles. The specific prescription is shown in Table 6:

[0183] Table 6. Peptide nanoparticles formulation and their particle size, potential, and encapsulation efficiency

[0184]

[0185] The prepared polypeptide complex nanoparticle aqueous solution is mixed with a lyophilized agent, and then freeze-dried using a freeze dryer (ChristAlphaLD plus, Germany) to prepare a lyophilized agent. The lyophilized agent can be trehalose or sucrose, and is stored in a refrigerator at 4°C for later use.

[0186] Example 3: Characterization of the polypeptide complex nanoparticles of the present invention

[0187] Nanoparticle morphology:Using EGFP-mRNA as a model mRNA, polypeptide complex nanoparticles were prepared according to the preparation method 1, preparation method 2, preparation method 3, or preparation method 4 and the corresponding prescriptions described in Example 2. The representative aqueous solution of polypeptide complex nanoparticles described in the present invention was tested for its nanoparticle morphology using a transmission electron microscope (model FEI Talos F200X). A copper grid without any dyeing was immersed in the freshly prepared aqueous solution of polypeptide complex nanoparticles and dried naturally at 25°C to obtain a sample. The test was performed, as shown in FIG. Figure 1 As shown, the results show that the polypeptide complex nanoparticles of the present invention have good dispersibility, regular or irregular spherical structure, and particle size ranges from 60nm to 120nm. Figure 1 shown.

[0188] Particle size and potential :Using EGFP-mRNA as a model mRNA, polypeptide complex nanoparticles were prepared according to the preparation method described in Example 2, and the dynamic light scattering particle size (size), surface potential (Zeta Potential) and polydispersity (PDI) of the polypeptide complex nanoparticles were tested at 25°C using a Malvern Zetasizer Nano ZSE. The results are shown in Tables 3 to 6, and the results show that the particle size of the polypeptide complex nanoparticles of the present invention ranges from 56nm to 273nm, has good dispersibility, and the surface charge of the nanoparticles is between -15mV and 5mV.

[0189] Encapsulation efficiency :Using FLuc-mRNA as the model mRNA, polypeptide complex nanoparticles were prepared according to the preparation method described in Example 2, and the mRNA encapsulation efficiency of each formulation was determined using the Quant-iT RiboGreen RNA detection kit (ThermoFische Company). For specific methods, refer to the instructions of the kit. The brief processing method of the present invention is as follows: using a low-temperature high-speed centrifuge to centrifuge each formulation at 4°C, 20000rpm for 2h, collecting the supernatant and quantifying its volume with a pipette, recorded as V1; using the Quant-iTRiboGreen RNA detection kit to measure the concentration of mRNA in the supernatant, recorded as C1; adding 25ul of chromatographically pure DMSO to dissolve the precipitate after centrifugation, and continuing to add 0.9% physiological saline injection to mix evenly, after standing at 25°C for 2h, recording the total volume V2 and quantifying it with Quant-iT RiboGreen The RNA detection kit was used to measure the concentration of mRNA, which was recorded as C2. The encapsulation rate of each prescription was calculated as follows: encapsulation rate = 100% - (V1C1) / (V1C1 + V2C2) × 100%. The results are shown in Tables 3 to 6. The prescriptions all had good encapsulation effects on mRNA, and the encapsulation rates were all above 98.0%.

[0190] Example 4: Detection of the ability of polypeptides to compress mRNA by agarose gel electrophoresis

[0191] Agarose gel with a mass volume ratio of 1% (agarose 0.4 g: 1×TAE buffer solution 40 ml) was prepared and microwaved twice to fully melt it. 4 μl SyBR Safe DNA Gel Stain dye (Lotno.1771519, Invitrogen, USA) was added to the agarose at a ratio of 1:10000. After fully mixing, it was poured into the corresponding gel slot (15-well card slot) and cooled for 20 minutes for use.

[0192] Preparation method of mRNA positive control group: Take EGFP-mRNA as model mRNA, add 1μl mRNA solution with a concentration of 100ng / μl (i.e. 100ng), then add 9μl nuclease-free ultrapure water to make the system volume up to 10μl, and finally add 2μl 6*Loading Buffer and mix well.

[0193] Sample group configuration method: According to the different mass ratios of polypeptide and mRNA, 1μl of mRNA solution with a concentration of 100ng / ul (i.e. 100ng) was added to the polypeptide solution (1μg / μl) and mixed, and then nuclease-free ultrapure water was added to make the system volume up to 10μl. After mixing for 10 minutes, 2μl of 6*Loading Buffer was added to each sample and mixed. After the samples were mixed, 12μl of the system was added to each well, and the gel was run for 25 minutes using an 80V voltage electrophoresis instrument and observed using a gel imager. The experimental results are shown in the figure. Figure 2 shown.

[0194] Conclusion: The minimum mass ratio (peptide: mRNA) for each polypeptide to ensure complete compression of mRNA is: Seq.05 is 4, Seq.12 is 4, Seq.46 is 2, Seq.47 is 2, Seq.49 is 16, and Seq.53 is 4.

[0195] Example 5: In vitro cell transfection experiment and cytotoxicity study of polypeptide complex nanoparticles

[0196] Cell transfection with mRNA (using FLuc-mRNA as model mRNA): DC2.4 cell suspension in logarithmic growth phase was cultured at 4×10 4The cells were divided into 96-well plates at a density of 1 cell per well and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the FLuc-mRNA with a concentration of 1 μg / μl was diluted to 0.1 μg / μl with nuclease-free ultrapure water, and the FLuc-mRNA was taken to prepare polypeptide complex nanoparticles according to the preparation methods of different prescriptions described in Example 2, and then diluted to 88 μl of a polypeptide nanoparticle composition mixture containing 10 ng / μl FLuc-mRNA with nuclease-free ultrapure water. After standing for 10 minutes, 20 μl per well was added to each well of a 96-well plate containing 180 μl of opti-MEM culture medium, and each sample was repeated 4 wells. After 4 hours of administration, the culture medium in the 96-well plate was replaced with complete culture medium and aspirated. After culturing for 24 hours, the complete medium was aspirated and rinsed with PBS. 100 μl of D-Luciferin working solution (working concentration was 250 μg / ml) was added to each 96-well plate and cultured in a 37°C incubator for 5 minutes. The fluorescence expression intensity of FLuc-mRNA was tested by imaging with an Omega-FLuostar microplate reader. The results are shown in Figure 3 shown.

[0197] Cytotoxicity assay: DC2.4 cells in logarithmic growth phase were suspended at 4×10 4 The cells were divided into 96-well plates at a density of 1 cell per well and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the FLuc-mRNA with a concentration of 1 μg / μl was diluted to 0.1 μg / μl with nuclease-free ultrapure water, and the FLuc-mRNA was taken to prepare polypeptide complex nanoparticles according to the preparation methods of different prescriptions described in Example 2, and then diluted to 88 μl of a polypeptide nanoparticle composition mixture containing 10 ng / μl FLuc-mRNA with nuclease-free ultrapure water. After standing for 10 minutes, 20 μl per well was added to each well of a 96-well plate containing 180 μl of opti-MEM culture medium, and each sample was repeated 4 wells. After 4 hours of administration, the culture medium in the 96-well plate was replaced with complete culture medium and aspirated. Continue to culture for 48 hours, aspirate the complete medium, and rinse three times with PBS. Use the cell wells without prescription as negative controls, and the CCK-8 medium wells without cells as blank controls. Add 90μl serum-free medium and 10μl CCK-8 solution to each well, and continue to incubate in the incubator for 2 hours. Use Omega-FLuostar microplate reader to measure the absorbance at 450nm. Cell viability calculation formula:

[0198] Cell viability*%=[A(drug added)-A(blank)] / [A(no drug added)-A(blank)]×100%;

[0199] A (drug addition): absorbance of each well with DC2.4 cells, prescription solution, and CCK-8 solution added;

[0200] A (blank): absorbance of each well with only CCK-8 solution added;

[0201] A (no drug added): absorbance of each well with DC2.4 cells and CCK-8 solution added;

[0202] *Cell viability: cell proliferation activity or cytotoxic activity.

[0203] The results are as follows Figure 4 shown.

[0204] Conclusion: The results show that the cell survival rate is above 90%, indicating that the peptide complex nanoparticles have no obvious cytotoxicity and have good biocompatibility, and can be used for subsequent animal experiments. Cell transfection DNA (Luc-pDNA as model DNA): DC2.4 cell suspension in logarithmic growth phase was cultured at 4×10 4 The cells were divided into 96-well plates at a density of 1 cell per well and placed in a 37°C, 5% CO2 incubator for static culture. After 24 hours, the Luc-pDNA with a concentration of 1 μg / μl was diluted to 0.1 μg / μl with nuclease-free ultrapure water. Luc-pDNA was taken to prepare polypeptide complex nanoparticles according to the preparation methods of different prescriptions described in Example 2, and then diluted to 88 μl of a polypeptide nanoparticle composition mixture containing 15 ng / μl Luc-pDNA with nuclease-free ultrapure water. After standing for 30 minutes, 20 μl per well was added to each well of a 96-well plate containing 180 μl of opti-MEM culture medium, and each sample was repeated in 4 wells. 4 hours after administration, the culture medium in the 96-well plate was replaced with complete culture medium. Continue to culture for 24 hours, aspirate the complete medium, add 100μl of D-Luciferin solution with a working concentration of 250μg / ml to each 96-well plate, continue to culture in a 37℃ incubator for 5 minutes, and finally use Omega-FLuostar microplate reader to image and test the fluorescence expression intensity of Luc-pDNA. Repeat the test every 24 hours. After each test, aspirate the medium containing D-Luciferin, add fresh complete medium and continue to culture for 24 hours. Then add D-Luciferin test and repeat for three days. The results are as follows Figure 5 As shown in the figure, the horizontal axis represents different prescriptions, and the vertical axis represents the relative fluorescence intensity of Luc-pDNA expressed at the same dose after 24h, 48h, and 72h of transfection. Figure 5 shown.

[0205] Conclusion: Figure 5As shown, the polypeptide complex nanoparticles encapsulating Luc-pDNA showed good expression at the cell level, with the highest expression on the second day and decreasing from the third day, among which Rp.01, Rp.27, and Rp.34 were better than other prescriptions.

[0206] Example 6: Small animal fluorescence imaging to detect transfection of peptide complex nanoparticles in mice

[0207] Three female BALB / c mice were included in each group. FLuc-mRNA was used as a model mRNA, and a polypeptide complex nanoparticle formulation containing FLuc-mRNA was prepared by the method of the present invention. In the experimental group, 50 μl of the polypeptide complex nanoparticle formulation containing 5 μg FLuc-mRNA was injected into each mouse using an insulin needle. The administration method of the prescription Rp.01, Rp.07, Rp.11, Rp.12, Rp.13, Rp.17, Rp.20, Rp.22, and Rp.24 groups was subcutaneous injection, and the injection site was the subcutaneous part of the mouse back; the administration method of the prescription Rp.33, Rp.36, Rp.37, and Rp.42 groups was intraperitoneal injection; the administration method of the other prescription groups was intramuscular injection, and the injection site was the mouse thigh muscle. NC represents the blank control group, and 50 μl PBS buffer was injected intramuscularly with an insulin needle. Six hours after administration, take an appropriate amount of substrate D-Luciferin, dilute it with PBS to prepare a solution with a concentration of 25 mg / ml, keep it away from light, and inject 125 μl of substrate into each mouse intraperitoneally. Place the mouse in a small animal anesthesia box, open the ventilation valve, and release isoflurane to anesthetize the mouse. 5 minutes after substrate injection, use a small animal in vivo imaging system (PerkinElmer, IVIS Lumina SeriesⅢ) to perform whole-body in vivo imaging bioluminescence image detection on the mouse. Among them, the prescription Rp.33, Rp.36, Rp.37, and Rp.42 groups took bioluminescent images of the mouse abdomen, and the other prescription groups took bioluminescent images of the mouse back. The results are as follows Figure 6 As shown, one representative mouse was selected from each group, and the polypeptide complex nanoparticle formulations in the experimental group showed luciferase expression in whole-body in vivo imaging. The greater the fluorescence intensity, the more luciferase was expressed.

[0208] Conclusion: If Figure 6 As shown, the polypeptide complex nanoparticles of each experimental group encapsulating FLuc-mRNA have good luciferase expression in mice, and intraperitoneal injection, subcutaneous injection and intramuscular injection are all effective. In the experimental group prescriptions, Rp.27, Rp.33, and Rp.41 of luciferase are better than other prescriptions.

[0209] Example 7: Evaluation of humoral immune effects of polypeptide complex nanoparticles in mice

[0210] The SARS-CoV-2 S-mRNA was used as a model mRNA. The SARS-CoV-2 S-mRNA was provided by Hongene Biotech Corporation. The nucleotide sequence of the SARS-CoV-2 S-mRNA (cap1 structure, N1-me-pseudo U modified) is shown in the S-mRNA in the sequence list.

[0211] The specific information of S-mRNA stock solution is as follows:

[0212] Product Name: COVID-19 Spike Protein, Full Length-mRNA;

[0213] Product description: 4088 nucleotides in length;

[0214] Modifications: Fully substituted with N1-Me-pseudo UTP;

[0215] Concentration: 1.0mg / ml;

[0216] Storage environment: 1mM sodium citrate, pH 6.4;

[0217] Storage requirements: -40℃ or below.

[0218] Experimental process:

[0219] Step 1: First immunization of mice: On day 0, 5-6 week old female BALB / c mice were divided into 9 groups (5 mice in each group) and injected intramuscularly with 75 μl PBS (blank control), a combination of 5 μg naked S-mRNA and 5 μg S protein (positive control), and 75 μl of polypeptide complex nanoparticles Rp.21, Rp.25, Rp.27, Rp.41, Rp.01, and RP.08 loaded with 5 μg S-mRNA.

[0220] Step 2: First serum collection: On day 28, blood was collected from the outer canthus of the mice. After the serum coagulated at 4°C for 1 hour, it was centrifuged at 5000×g for 5 minutes at 4°C, and the supernatant was taken. It was then centrifuged at 10000×g for 5 minutes at 4°C, and the supernatant was added to eight consecutive PCR tubes and aliquoted and stored at -20°C for later use.

[0221] Step 3: Second immunization of mice: On the 28th day, after blood was collected from the outer canthus of the mice, 75 μl of PBS (blank control), a combination of 5 μg naked S-mRNA and 5 μg S protein (positive control), and 75 μl of peptide complex nanoparticles Rp.21, Rp.25, Rp.27, Rp.41, Rp.01, and RP.08 loaded with 5 μg S-mRNA were injected intramuscularly. The first immunization process was repeated.

[0222] Step 4: Second serum collection: 21 days after the second immunization, blood was collected from the outer canthus of the mice. After the serum coagulated at 4°C for 1 hour, it was centrifuged at 5000×g (5000 times the acceleration of gravity) for 5 minutes at 4°C, and the supernatant was taken. It was then centrifuged at 10000×g for 5 minutes at 4°C, and the supernatant was added to eight consecutive PCR tubes for aliquoting and stored at -20°C for later use.

[0223] Step 5: ELISA to detect serum IgG content: Dilute the S protein in PBS, coat the ELISA plate with 100μl of diluent (containing 1μg S protein) per well, and coat at 4℃ for 6h. Discard the liquid in the plate, add 200μl PBST to each well and wash the plate 3 times, then add 200μl PBS blocking solution containing 5% BSA to each well and block at 25℃ for 2h. Discard the blocking solution, wash the plate once with 200μl PBST per well, add 100μl of serum diluted 200 times with PBS, and incubate at 25℃ for 2h. Discard the serum, wash the plate 3 times with 200μl PBST per well, then add 100μl of antibody (antibody and PBS diluted at a ratio of 1:1000) to each well, and incubate at 25℃ for 1h. Discard the antibody, wash the plate 3 times with 200μl PBST per well, add 50μl TMB colorimetric solution per well to react in the dark, wait until the positive control well turns dark blue or reacts for 10 minutes, then add 50μl 2M sulfuric acid to each well to terminate the reaction, and measure the optical density at 450nm and 630nm with a microplate reader, and calculate the OD value difference to reflect the level of anti-S protein IgG in the serum. Figure 7 shown.

[0224] Summary: The results show that the OD values ​​corresponding to the prescription Rp14 were higher than those of the control group after two immunizations, indicating that the nanoparticles of this prescription have stronger serum conversion efficiency and humoral immune activation function.

[0225] Step 6: ELISA to detect serum IgG titer: Dilute the S protein in PBS, coat the ELISA plate with 100 μl of diluent (containing 1 μg of S protein) per well, and coat at 4°C for 6 hours. Discard the liquid in the plate, add 200 μl of PBST to each well to wash the plate once, then add 200 μl of PBS blocking solution containing 5% BSA to each well and block on a shaker at 25°C for 2 hours. Discard the blocking solution, wash the plate 3 times with 200 μl PBST per well, then add serum diluted 50, 250, 1250, 6250, 31250, 156250, 781250, 3906250 times with PBS at a ratio of 1:3, and incubate at 25°C for 2 hours. Discard the serum, wash the plate 3 times with 200μl PBST per well, add 100μl antibody (antibody and PBS diluted at a ratio of 1:1000) to each well, and incubate on a shaker at 25℃ for 1h. Discard the antibody, wash the plate 3 times with 200μl PBST per well, add 50μl TMB colorimetric solution to each well to react in the dark, wait until the positive control well turns dark blue or reacts for 10 minutes, then add 50μl 2M sulfuric acid to each well to terminate the reaction, and detect the optical density at 450nm and 630nm with an enzyme reader. The results are as follows Figure 8 shown.

[0226] Summary: The present invention takes twice the average OD value of the PBS group as the baseline. When the OD value of the Rp.08 group was diluted to 3906250 times, it was still twice higher than the baseline, indicating that the prescription Rp.08 has a strong serum conversion efficiency and humoral immune activation function.

[0227] Example 8: Gene transfection kit

[0228] The gene transfection kit is a multi-purpose transfection reagent that can be composed of any one of the prescriptions of the present invention, and can provide efficient transfection in various adherent and suspension cell lines. It is suitable for all common cell lines and many difficult-to-transfect cell lines, and can be used in culture media containing or not containing serum. The kit of the present invention is used to transfect mammalian cells in a 96-well cell culture plate. The specific steps are:

[0229] 1. One day before transfection, add 1×10 4 Up to 10×10 4 The cells were inoculated in a 96-well cell culture plate with 200 μl of culture medium in each well, so that the cell growth density reached more than 80% during transfection.

[0230] 2. For each transfection sample, prepare the following complexes:

[0231] a. Dilute 200 ng DNA or RNA to 15 μl with sterile nuclease-free water and mix gently;

[0232] b. Gently mix the transfection reagent according to the prescribed ratio before use, and then dilute the appropriate amount to 15μl with sterile nuclease-free water;

[0233] c. Gently mix the diluted DNA or RNA according to the prescription ratio and the diluted transfection reagent (total volume = 30 μl), and incubate at 25° C. for 10 to 30 minutes to obtain nucleic acid-polypeptide complex nanoparticles.

[0234] 3. After rinsing the 96-well plate with PBS, add 170 μl of Opti-MEM culture medium to each well, and then add 30 μl of nucleic acid-polypeptide complex nanoparticles. The final volume of the culture medium is 200 μl.

[0235] 4. Place the cells in a carbon dioxide incubator and incubate them. After 4 hours, replace the original culture medium with complete culture medium and continue to incubate in the carbon dioxide incubator for 12 to 72 hours. Finally, detect the expression level of the nucleic acid.

[0236] All references disclosed herein, including patent documents, are incorporated by reference.

[0237] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide compound, the amino acid sequence of the polypeptide compound is: Seq ID NO:

49.

2. A polypeptide complex nanoparticle, comprising: a) the polypeptide compound according to claim 1; and b) Nucleic acids, The nucleic acid is a nucleic acid drug.

3. The polypeptide complex nanoparticle according to claim 2, comprising: The nucleic acid and amino acid sequence are a polypeptide compound of Seq.49 and auxiliary materials, wherein the auxiliary materials are 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol; the mass ratio of the nucleic acid and amino acid sequence is Seq.49, 1,2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1,2-distearoyl-sn-glycero-3-phosphocholine and cholesterol is 10:300:8:16:

31.

4. A nucleic acid vaccine, comprising the polypeptide complex nanoparticles according to any one of claims 2-3.

5. Use of the polypeptide complex nanoparticles according to any one of claims 2 or 3 in the preparation of nucleic acid drugs or gene transfection kits.

Images