WO2022048619A1 - Peptide and peptide complex nanoparticle, nucleic acid vaccine and application thereof - Google Patents

Peptide and peptide complex nanoparticle, nucleic acid vaccine and application thereof Download PDF

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WO2022048619A1
WO2022048619A1 PCT/CN2021/116359 CN2021116359W WO2022048619A1 WO 2022048619 A1 WO2022048619 A1 WO 2022048619A1 CN 2021116359 W CN2021116359 W CN 2021116359W WO 2022048619 A1 WO2022048619 A1 WO 2022048619A1
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seq
peptide
nucleic acid
rna
peptide complex
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PCT/CN2021/116359
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French (fr)
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Longgui ZHANG
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Shenzhen Longuide Biopharma Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455—Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • the present invention relates to the field of drug delivery, and particularly to a peptide and a peptide complex nanoparticle, a nucleic acid vaccine and applications thereof.
  • Gene transfection is a technology for transferring or transporting nucleic acids with biological functions into cells and making nucleic acids maintain the biological functions thereof in the cells.
  • a gene vector is a tool for introducing exogenous therapeutic genes into biological cells.
  • Nucleic acid vaccine is a new type of vaccines that have been developed in recent years. As a new response option, the nucleic acid vaccine is to introduce nucleic acid encoding antigen protein into cells and synthesize the protein through a cell expression system, so as to induce specific immune response. Although most cells can spontaneously take up nucleic acids, the efficiency is very low and the cells are saturated at low doses.
  • RNA enzymes RNases
  • DCs dendritic cells
  • CPPs Cell penetrating peptides
  • HAV-1 TAT human immunodeficiency virus-1 transcription activator
  • CPPs net positive or electroneutral charge, hydrophilicity and hydrophobicity (amphipathy) ; high efficiency of membrane penetration delivery; low cytotoxicity; no cell type restriction; and the ability to introduce different bioactive substances into cells by chemical binding or gene fusion, making them potentially versatile targeted drug carriers.
  • the specific transmembrane mechanisms vary among CPPs, and particular amino acid sequences have been found to bind to mRNA and to interfere with and reduce cell membrane stability, thereby carrying bioactive substances across the cell membrane, such as the arginine-alanine-leucine-alanine residue (RALA) sequence (Pardi et al. Curt Opin in Immunol. 2020, 65: 14 -20) .
  • RALA arginine-alanine-leucine-alanine residue
  • the present invention addresses the shortcomings of existing delivery systems, synthesizes non-naturally occurring peptides, and prepares peptide complex nanoparticles to provide improved gene vector for mRNA delivery and nano-delivery solutions suitable for animal vaccine or human mRNA drug development.
  • the present invention provides a peptide compound for nucleic acid drug delivery.
  • the present invention provides a peptide complex nanoparticle containing the peptide compound.
  • the present invention provides an application of a peptide complex nanoparticle in nucleic acid delivery in vitro and in vivo.
  • the present invention provides a nucleic acid vaccine containing the peptide complex nanoparticles.
  • the present invention provides a use of the peptide complex nanoparticles in preparing medicines or kits.
  • the present invention provides a peptide compound having a following structure of general formula I:
  • x is an integer from 1 to 25 and y is an integer from 0 to 10;
  • (Xaa) x is a peptide segment composed of any amino acid.
  • 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)
  • x is the number of amino acids, with x being 1 to 20.
  • (Xaa) x is Arg.
  • (Xaa) x is (Xa′a′) n (Arg) 1-10 (Xa′a′) n , where Xa′a′ is selected from 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.
  • Xaa consists of (Arg) 1-10 , Trp (W) , and/or Cys (C) .
  • Xaa consists of (Arg) 1-10 , Trp (W) , Cys (C) , His (H) , and/or Pro (P) , with Trp (W) , Cys (C) , His (H) , and/or Pro (P) either preceding or following (Arg) 1- 10 , or interspersed in one or several (Arg) 1-10 in between.
  • a sequence of Arg (R) , Trp (W) , Cys (C) , Lys (K) , Leu (L) , Phe (F) , Pro (P) or His (H) in amino acid sequences is not limited.
  • (Yaa) y is a peptide segment consisting of any amino acid; in some embodiments, Yaa is selected from at least one of Arg (R) , Trp (W) , Phe (F) , or Cys (C) , and y is the number of amino acids, with y being 0 to 10,.
  • the x can be1, 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.
  • the y can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • an amino acid sequence of the peptide 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.
  • the amino acid sequence of the peptide compound is: Seq. 05, Seq. 12, Seq. 46, Seq. 47, Seq. 49 or Seq. 53.
  • the general formula (I) is at least 50%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 20%; in some embodiments, the general formula (I) is at least 75%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 50%; in some embodiments, the general formula (I) is at least 90%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 100%; and
  • the general formula (I) is at least 90%similar to any one of RRRRRWCRVQPTESIVR, RRRRRWFCRVQPTESIVR, FCRWCRRVQPTESIVRRCWRCF, FCRWCRRVQPTESIVCWRRRCF, HKRWCRRWCRVQPTESIVRC or WCRRRVQPTESIVRRRWC.
  • the peptide of general formula (I) includes 10-35 amino acids, which is characterized in that the peptide improves the delivery of nucleic acid molecules into the cell by at least 10%, in some embodiments, the peptide of general formula (I) includes 10-35 amino acids, which is characterized in that the peptide improves the delivery of nucleic acid molecules into the cell by between about 50%and about 100%; and in some embodiments, the peptide of general formula (I) includes 10-35 amino acids, which is characterized in that the peptide improves the delivery of nucleic acid molecules into the cell by between about 75%and about 500%.
  • the present invention provides novel non-naturally occurring peptides with functions such as compressing and protecting nucleic acids from degradation and facilitating penetration of nucleic acids through cell membranes, as well as peptide complex nanoparticles containing the peptides, and methods for applying the peptide complex nanoparticles to gene transfection of cells in vivo and in vitro, and methods of applying the peptide complex nanoparticles to vaccine formulations.
  • the present invention provides a peptide complex nanoparticle.
  • a peptide complex nanoparticle which includes:
  • a peptide complex nanoparticle which includes:
  • the nucleic acid may be chemically modified or unmodified DNA, single-stranded or double-stranded DNA, coding or non-coding DNA.
  • the nucleic acid is selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA primers, DNA probes, immunostimulatory DNA, aptamers, or any combination thereof.
  • the nucleic acid may be chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA.
  • the nucleic acid is selected from messenger RNA (mRNA) , oligonucleotides, viral RNA, replicon RNA, transfer RNA (tRNA) , ribosomal RNA (rRNA) , immunostimulatory RNA (isRNA) , micro RNA, small interfering RNA (siRNA) , small nuclear RNA (snRNA) , small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA decoy, antisense RNA, nuclease, or any combination thereof.
  • the nucleic acid is a chemically modified messenger RNA (mRNA) .
  • the nucleic acid sequences of the RNA may include all the nucleic acid sequences listed in patent US9254311B2, also include all the sequences listed in the long sequence appendix of the patent.
  • the RNA sequences described in the present invention can be obtained by the nucleic acid synthesis method listed in patent US9254311B2 or CN106659803A.
  • the peptide complex nanoparticles can encapsulate mRNA and allow its efficient introduction into different cell lines in vitro and can be efficiently transfected in vivo.
  • the peptide complex nanoparticle of the present invention can carry mRNA encoding an immunogenic peptide into cells and effectively release the mRNA to express antigens and effectively achieve immunotherapy or immunoprophylaxis.
  • the present invention provides novel non-naturally occurring peptides with functions such as compressing and protecting nucleic acids from degradation and facilitating penetration of nucleic acids through cell membranes, as well as peptide complex nanoparticles containing the peptides, and methods for applying the peptide complex nanoparticle to gene transfection of cells in vivo and in vitro, and methods of applying the peptide complex nanoparticle to vaccine formulations.
  • the present invention provides a peptide complex nanoparticle, which includes:
  • the auxiliary material may be selected from one or more of phospholipids, PEG lipids or PEG derivatives.
  • the lipid may be a naturally occurring or synthetic phospholipid or structural lipid.
  • the PEG derivative may be poloxamide, poloxamide derivative, poloxamer derivative or PEG lipid.
  • the poloxamide can be selected from at least one of the following poloxamide: or and The poloxamide derivatives are synthesized with reference to patent CN111285845B.
  • the poloxamer can be selected from at least one of the following poloxamer: 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 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.
  • the phospholipids can be selected from: 1, 2-distearoyl-sn-glycerol-3-phosphate choline (DSPC) , 1, 2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) , 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DLPC) , 1, 2-dimyristoyl-sn-glycerol-phosphate choline (DMPC) , 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC) , 1, 2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC) , 1, 2-bis-undecanoyl-sn-glycerol-phosphate choline (DUPC) , 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) , 1, 2-di
  • the structural lipids may be selected from: cholesterol (Chol) , stersterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, ⁇ -tocopherol, and mixtures thereof; and
  • the PEG lipid may be selected from any of the PEG lipids described in Patent Nos. CN111281981B, CN111315359A, CN111356444A, such as 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-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) , such as mPEG5000-C-CLS (PEG-CLS) , such as mPEG2000-DSPE (PEG-DSPE) .
  • DMG-PEG 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000
  • DMG-PEG mPEG5000-C-CLS
  • PEG-DSPE m
  • the 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) has the following structural formula:
  • the mPEG5000-C-CLS (PEG-CLS) has the following structural formula:
  • the mPEG2000-DSPE (PEG-DSPE) has the following structural formula:
  • a mass ratio of the nucleic acid to the peptide may be less than or equal to about 1 ⁇ 1. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 1 to about 1 ⁇ 52. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 48. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 40. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 32. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 24.
  • the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 16. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 10. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 toabout 1 ⁇ 8. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 5. In some embodiments, the mass ratio of the nucleic acid to the peptide is 1 ⁇ 2 to 1 ⁇ 4.
  • the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 1, 1 ⁇ 2, 1 ⁇ 3, 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.
  • an amino acid sequence of the peptide is Seq. 05, and the mass ratio of the nucleic acid to the peptide is less than or equal to 1 ⁇ 4. In some embodiments, the amino acid sequence of the peptide is Seq. 05, and the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 4 to about 1 ⁇ 52.
  • the amino acid sequence of the peptide is Seq. 12, and the mass ratio of the nucleic acid to the peptide is less than or equal to 1 ⁇ 4. In some embodiments, the amino acid sequence of the peptide is Seq. 12, and the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 4 to about 1 ⁇ 52.
  • the amino acid sequence of the peptide is Seq. 46, and the mass ratio of the nucleic acid to the peptide is less than or equal toabout 1 ⁇ 2. In some embodiments, the amino acid sequence of the peptide is Seq. 46, and the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 52.
  • the amino acid sequence of the peptide is Seq. 47, and the mass ratio of the nucleic acid to the peptide is less than or equal to about 1 ⁇ 2. In some embodiments, the amino acid sequence of the peptide is Seq. 47, and the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 52.
  • the amino acid sequence of the peptide is Seq. 49, and the mass ratio of the nucleic acid to the peptide is less than or equal to about 1 ⁇ 2. In some embodiments, the amino acid sequence of the peptide is Seq. 49, and the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 16 to about 1 ⁇ 52.
  • the amino acid sequence of the peptide is Seq. 53, and the mass ratio of the nucleic acid to the peptide is less than or equal to about 1 ⁇ 4. In some embodiments, the amino acid sequence of the peptide is Seq. 53, and the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 4 to about 1 ⁇ 52.
  • the mass ratio of the nucleic acid to the auxiliary arrangement may be less than or equal to about 1 ⁇ 2. In some embodiments, the mass ratio of the nucleic acid to the auxiliary arrangement 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 to about 1 ⁇ 800. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 500. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 50.
  • the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 33. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 10. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 , about 1 ⁇ 3, about 1 ⁇ 4, about 1 ⁇ 5 or about 1 ⁇ 6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 33 to about 1 ⁇ 400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 50 to about 1 ⁇ 800.
  • the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 50 to about 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 about 1 ⁇ 100 , about 1 ⁇ 200 or about 1 ⁇ 500. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2, the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 50 to about 1 ⁇ 800.
  • the auxiliary materials are PEG derivatives, phospholipids and structural lipids, and the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to 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 material is about 1 ⁇ 2 , about 1 ⁇ 3, about 1 ⁇ 4, about 1 ⁇ 5 or about 1 ⁇ 6.
  • the mass ratio of the nucleic acid to the peptide is about 1 ⁇ 2 to about 1 ⁇ 30
  • the auxiliary materials are PEG derivatives, phospholipids and structural lipids
  • the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 2 to about 1 ⁇ 6.
  • the auxiliary materials are PEG derivatives and phospholipids, and the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 33 to about 1 ⁇ 400 or about 1 ⁇ 33 to about 1 ⁇ 370. In some embodiments, the auxiliary materials are PEG derivatives and phospholipids, the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 33 to about 1 ⁇ 400 or about 1 ⁇ 33 to about 1 ⁇ 370, and the mass ratio of the PEG derivative and phospholipid is 32 ⁇ 1 to 700 ⁇ 1.
  • the auxiliary materials are PEG derivatives and phospholipids
  • the mass ratio of the nucleic acid to the auxiliary material is 1 ⁇ 2
  • the mass ratio of the nucleic acid to the auxiliary material is about 1 ⁇ 33 to about 1 ⁇ 400 or about 1 ⁇ 33 to about 1 ⁇ 370
  • the mass ratio of the PEG derivative and phospholipid is about 32 ⁇ 1 to 700 ⁇ 1.
  • a peptide complex nanoparticle composition which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 05 and auxiliary materials, the auxiliary materials are and lecithin , the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 05, and lecithin is about 1 ⁇ 2 ⁇ 322 ⁇ 1.
  • a peptide complex nanoparticle composition which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 49 and auxiliary materials, the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 49, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol is about 10 ⁇ 300 ⁇ 8 ⁇ 16 ⁇ 31.
  • a peptide complex nanoparticle composition which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 53 and auxiliary materials, the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 53, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol is about about 10 ⁇ 40 ⁇ 8 ⁇ 16 ⁇ 5.
  • a peptide complex nanoparticle includes at least one non-naturally occurring peptide and nucleic acid of the present invention.
  • a peptide complex nanoparticle includes at least one non-naturally occurring peptide, nucleic acid and a least one lipid or PEG derivative of the present invention.
  • the nucleic acid may be chemically modified or unmodified DNA, single-stranded or double-stranded DNA, coding or non-coding DNA.
  • the nucleic acid is selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA primers, DNA probes, immunostimulatory DNA, aptamers, or any combination thereof.
  • the nucleic acid may be chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA.
  • the nucleic acid is selected from messenger RNA (mRNA) , oligonucleotides, viral RNA, replicon RNA, transfer RNA (tRNA) , ribosomal RNA (rRNA) , immunostimulatory RNA (isRNA) , micro RNA, small interfering RNA (siRNA) , small nuclear RNA (snRNA) , small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA decoy, antisense RNA, nuclease, or any combination thereof.
  • the nucleic acid is a chemically modified messenger RNA (mRNA) .
  • the nucleic acid sequences of the RNA may include all the nucleic acid sequences listed in patent US9254311B2, also include all the sequences listed in the long sequence appendix of the patent.
  • the RNA sequences described in the present invention can be obtained by the nucleic acid synthesis method listed in patent US9254311B2 or CN106659803A.
  • the peptide complex nanoparticle may further include at least one pharmaceutically acceptable excipient.
  • the peptide complex nanoparticle of the present invention is stable in aqueous solution and can be contacted with human or animal cell tissues after formation, or can be stored for a period of time before contact with the cells or tissues.
  • the peptide complex nanoparticle is stable, and can be stored for 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. It should be understood that the storage period may be between any of these time periods for example between 31 minutes and 1 hour or between 1 hour and 24 hours.
  • the present invention provides an application of a peptide complex nanoparticle in nucleic acid delivery in vitro and in vivo.
  • the peptide complex nanoparticle can encapsulate mRNA and allow its efficient introduction into different cell lines in vitro and can be efficiently transfected in vivo.
  • the peptide complex nanoparticle of the present invention can carry mRNA encoding an immunogenic peptide into cells and effectively release the mRNA to express antigens and effectively achieve immunotherapy or immunoprophylaxis.
  • the present invention provides novel non-naturally occurring peptides with functions such as compressing and protecting nucleic acids from degradation and facilitating penetration of nucleic acids through cell membranes, as well as peptide complex nanoparticle containing the peptides, and methods for applying the peptide complex nanoparticle to gene transfection of cells in vivo and in vitro, and methods of applying the peptide complex nanoparticle to vaccine formulations.
  • the present invention provides a nucleic acid vaccine containing the pepfide complex nanoparticle of the second aspect.
  • a nucleic acid vaccine is provided, the nucleic acid vaccine includes the peptide complex nanoparticle of the second aspect.
  • the peptide complex nanoparticle may include at least one RNA.
  • the nucleic acid vaccine can be used for treating or preventing diseases.
  • the RNA includes at least coding RNA.
  • the encoding RNA may include RNA capable of encoding at least one coding region of at least one therapeutic protein, therapeutic peptide, immunogenic protein or immunogenic peptide.
  • the coding RNA is mRNA.
  • RNA vaccine with RNA (such as messenger RNA (mRNA) ) as the core and peptide complex nanoparticle as the delivery agent, which can safely induce the naturally occurring specific immune system of the body to produce almost any protein of interest or its fragment, including infectious pathogen vaccines such as bacteria and viruses and tumor vaccines.
  • infectious pathogen vaccines such as bacteria and viruses and tumor vaccines.
  • RNA is modified.
  • the nucleic acid vaccine disclosed by the invention can be used for inducing immune responses against infectious pathogens or cancers, including cellular immune responses and humoral immune responses, without the risk of insertion mutagenesis that may result.
  • nucleic acid vaccines with peptide complex nanoparticle as delivery agents can be used in various disease types.
  • the nucleic acid vaccine can be used for preventing and/or treating infectious pathogens or cancers of various metastatic stages or degrees.
  • a nucleic acid vaccine in some embodiments of the present invention, includes the peptide complex nanoparticle of the second aspect; the peptide complex nanoparticle includes at least one RNA; the RNA is messenger RNA (mRNA) ; the messenger RNA (mRNA) can safely direct the body′scellular mechanisms to produce almost any protein of interest, from natural proteins to antibodies and other completely novel protein constructs that can be therapeutically active inside and outside the cell.
  • mRNA messenger RNA
  • mRNA messenger RNA
  • the nucleic acid vaccine is available in a variety of contexts depending on 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 advantage of the nucleic acid vaccine is that it produces a much larger antibody titer compared to commercially available antiviral treatment and reacts earlier.
  • RNA vaccines like mRNA polynucleotides, are better designed to produce appropriate protein conformation by translocation when the RNA vaccine assigns a natural cellular mechanism.
  • the nucleic acid vaccine provides a template for the cellular system to express protein antigens in a more natural manner.
  • a nucleic acid vaccine in some embodiments of the present invention, includes the peptide complex nanoparticle of the second aspect; the peptide complex nanoparticle includes at least one RNA;
  • the nucleotide sequence of the RNA is a nucleotide sequence encoding an antigen of any pathogen.
  • the RNA is mRNA.
  • the RNA is mRNA whose nucleotide sequence encodes the S spike protein of novel coronavirus SARS-CoV-2.
  • a nucleic acid vaccine in some embodiments of the present invention, includes the peptide complex nanoparticle of the second aspect; the peptide complex nanoparticle contains artificially synthesized pathogen antigenic peptides. In some embodiments, the antigenic peptide is fused with other peptides that enhance transfection and delivery efficiency and/or enhance immune response.
  • the dosage form of the nucleic acid vaccine can be injection, tablet, inhalation formulation, suppository, eye drop or suspension, etc..
  • the nucleic acid vaccine of the present invention can be administered by any route that produces a therapeutically effective result.
  • routes include, but are not limited to, intradermal, subcutaneous, intraperitoneal, oral, intramuscular, intranasal, intraocular, upper respiratory, intravenous, vaginal, rectal administration.
  • the mRNA vaccine of the present invention is administered by injection.
  • the present invention provides a use of the peptide complex nanoparticle of the second aspect in preparing medicines or kits.
  • a use of the peptide complex nanoparticle of the second aspect in preparing medicines or kits is provided.
  • a use of the peptide complex nanoparticle of the second aspect preparing medicines for the prevention, treatment and/or amelioration of a disease selected from the group consisting of: cancer or rumor diseases, infectious diseases, autoimmune diseases, allergic reaction or allergic disease, monogenic hereditary diseases, or a general genetic disease, diseases with genetic background and typically caused by identified genetic defects and inherited according to Mendelian law, cardiovascular diseases, neuronal diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal diseases, connective tissue diseases, rum, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases and ear diseases.
  • a disease selected from the group consisting of: cancer or rumor diseases, infectious diseases, autoimmune diseases, allergic reaction or allergic disease, monogenic hereditary diseases, or a general genetic disease, diseases with genetic background and typically caused by identified genetic defects and inherited according to Mendelian law, cardiovascular diseases, neuronal diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal diseases, connective tissue diseases, rum, immunodefic
  • infectious diseases may include viral infectious diseases, bacterial infectious diseases or protozoological infectious diseases.
  • FIG. 1 shows a transmission electron microscopic diagram of the peptide complex nanoparticle of Example III; where A represents recipe Rp. 05, B represents recipe Rp. 28, C represents recipe Rp. 43, and the white scale is 200 nm.
  • FIG. 2 shows agarose gel electrophoresis results of the peptide complex nanoparticles of Example IV; where mRNA refers to mRNA positive control group, and 1, 2, 4, 8, 16, 32 and 64 refer to the mass ratio of the peptide to mRNA is 1 ⁇ 1, 2 ⁇ 1, 4 ⁇ 1, 8 ⁇ 1, 16 ⁇ 1, 32 ⁇ 1 and 64 ⁇ 1; and the minimum mass ratio of each peptide to ensure that mRNA is fully compressed is: 4 for Seq. 05, 4 for Seq. 12, 2 for Seq. 46, 2 for Seq. 47, 16 for Seq. 49 and 4 for Seq. 53.
  • FIG. 3 shows a transfection of FLuc-mRNA peptide complex nanoparticles in DC2.4 cells in Example V; where the abscissa represents peptide nanoparticle compositions of different prescriptions, and the ordinate represents a relative fluorescence intensity expressed after transfection of the peptide nanoparticle compositions containing the same dose of FLuc-mRNA for 24 hours.
  • FIG. 4 shows a survival rate of DC2.4 cells treated with different prescriptions in Example V; where the abscissa represents different prescriptions of peptide complex nanoparticles, and the ordinate represents cell activity, the higher the cell activity, the smaller the cytotoxicity.
  • FIG. 5 shows a transfection of Luc-pDNA peptide complex nanoparticles in DC2.4 cells in Example V; the abscissa represents different prescriptions, and the ordinate shows a relative fluorescence intensity expressed by DC2.4 cells at 24h, 48h and 72h after transfection of Luc-pDNA with the same dose.
  • FIG. 6 shows an expression of luciferase of peptide complex nanoparticles in mice detected by IVIS (In Vivo Imaging Systems) in Example VI.
  • FIG. 7 shows a serum IgG antibody level of mice immunized with peptide complex nanoparticles in Embodiment VII; the abscissa represents the 28th and 49th days after the first immunization of different prescriptions, and the ordinate represents the difference of OD value of optical density at two wavelengths, OD value is an index to judge the level of IgG antibody in serum, reflecting the level of anti-S protein IgG in serum.
  • FIG. 8 shows a serum IgG antibody titer of mice immunized with peptide complex nanoparticles in Embodiment VIII; the abscissa represents different dilution times of serum after 49 days after the first immunization with different prescriptions, and the ordinate represents the difference of OD value (optical density value) at two wavelengths.
  • 2x Baseline double of background
  • the cut-off value is used as the cut-off value to distinguish positive and negative results, and the maximum dilution of the OD value higher than this value.
  • the terms “about” as used herein represent an amount close to the stated amount that still performs the desired function or achieves the desired result.
  • the terms “about” may refer to an amount that is within less than 10%of, within less than 5%of, within less than 1%of, within less than 0.1%of, and within less than 0.01%of the stated amount.
  • peptide refers to a polymer of amino acid residues (natural or non-natural) that are in many cases linked together by peptide bonds.
  • the term refers to proteins, peptides, and peptides of any size, structure, or function.
  • Peptides may be single molecules or may be multimolecular complexes such as dimers, trimers, or tetramers. They can also contain single-chain or multi-chain peptides such as antibodies or insulin, and can be associated or linked. The most common disulfide bonds are found in multi-chain peptides.
  • the term peptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids.
  • protein refers to a polymer consisting essentially of any of the 20 amino acids.
  • peptide is usually used to refer to relatively large peptides and “peptide” is usually used to refer to small peptides, the use of these terms in the field overlaps and varies.
  • peptide is sometimes used interchangeably.
  • hydrophilic means soluble in water under specific conditions, including readily soluble in water, soluble in water and slightly soluble in water.
  • hydrophobic means poorly soluble in water under certain conditions.
  • amino acid generally refers to naturally occurring or synthetic amino acids, as well as amino acid analogues and amino acid mimetics that act in a similar manner to naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code and those subsequently modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphate serine.
  • Amino acid analogues refer to compounds having the same basic chemical structure as naturally occurring amino acids (i.e., alpha carbon bound to hydrogen, carboxyl, amino and R groups) , such as homoserine, n-leucine, methionine sulfoxide, methionine and methyl sulfonium.
  • amino acid mimetics refer to compounds having a structure different from the general chemical structure of amino acids but function in a similar way to naturally occurring amino acids.
  • amino acid may refer to amino acids or their derivatives (e.g., amino acid analogues) and their D and L forms.
  • amino acids examples 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 acid, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, N-acetylcysteine.
  • Kit means a transfection, DNA, RNAi or other bioactive (e.g., protein or anion molecule) delivery or protein expression or knockdown kit that includes one or more reagents of the present invention or mixtures thereof.
  • the kit may include one or more non-naturally occurring peptides described herein, or optionally in combination with one or more lipids or PEG derivatives.
  • that peptide and lipid agent may be provided in the form of a single formulation.
  • the complex material and peptide may be provided separately, together with instructions instructing the user to combine the reagents in use.
  • Such kits may include carrying devices that are partitioned to hold one or more container devices (e.g., vials, test tubes, etc.
  • 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..
  • 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.
  • the media, methods, kits and compositions of the present invention are suitable for monolayer or suspension culture, transfection and cultivation of cells and for expression of proteins in monolayer or suspension cultured cells.
  • the media, methods, kits and compositions of the present invention are used for suspension culture, transfection and cultivation of cells, and for expression of protein products in suspension cultured cells.
  • Immune response the immune response can typically either 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) .
  • specific or adaptive immune response a specific response of the adaptive immune system to a specific antigen
  • non-specific or innate immune response a non-specific response of the innate immune system
  • Vaccine is typically understood as a prophylactic or therapeutic substance that provides at least one antigen or antigenic function.
  • the antigen or antigen function can stimulate the adaptive immune system of the body to provide an adaptive immune response.
  • mRNA for providing antigen can typically be mRNA with at least one open reading frame, which can be translated by cells or organisms provided with the mRNA.
  • the translated product is a peptide or protein which can be used as an antigen, preferably as an immunogen.
  • the product can also be a fusion protein composed of more than one immunogen, for example, a fusion protein composed of two or more epitopes, peptides or protein, wherein the epitopes, peptides or protein can be connected by a connecting sequence.
  • nucleic acid refers to any DNA or RNA molecule and is used synonymously with “polynucleotide” .
  • nucleic acid or nucleic acid sequence preferably further includes regulatory and/or other sequences that allow their expression and/or stability in a suitable host (e.g., human) , i.e., transcription and/or translation of the nucleic acid sequence encoding a particular protein or peptide.
  • a suitable host e.g., human
  • Peptides are polymers of amino acid monomers. Usually, 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 present invention, the peptide may for example contain less than 50 monomer units. Longer peptides may also be referred to as peptides and typically have 50 to 600 monomer units more specifically 50 to 300 monomer units. (Fotin-Mleczek Mariola et al., CN108064176A)
  • the pharmaceutically effective amount in the context of the present invention, is typically understood as an amount sufficient to induce an immune response or trigger a desired therapeutic effect. (Fotin-Mleczek Mariola et al., CN108064176A)
  • Protein proteins typically consist of one or more peptides and/or peptides that fold into a three-dimensional form and promote a biological function. (Fotin-Mleczek Mariola et al., CN108064176A)
  • RNA the chemical synthesis of relatively short segments of an oligonucleotide having a defined chemical structure provides a fast and inexpensive way to obtain a customized oligonucleotide of any desired sequence.
  • enzymes only synthesize DNA and RNA in the 5′ to 3′ direction
  • chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite direction (i.e., 3′ to 5′) .
  • this process is carried out in the form of solid phase synthesis using phosphoramidite methods and phosphoramidite structural units derived from protected nucleotides (A, C, G and U) or chemically modified nucleotides. (Fotin-Mleczek Mariola et al., CN108064176A)
  • the structural units are sequentially coupled to the growth oligonucleotide chain in the solid phase in the order required by the product sequence in a fully automated process.
  • the product is released from the solid phase to the solution, deprotected and collected.
  • the presence of side reactions imposes a practical limit on the length of the synthesized 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 with high purity. (Fotin-Mleczek Mariola et al., CN108064176A)
  • RNA in vitro transcription the term “RNA in vitro transcription” or “in vitro transcription” relates to the process in which RNA is synthesized (in vitro) in a cell-free system.
  • DNA in particular plasmid DNA
  • RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which is preferably a linearized plasmid DNA template according to the present invention.
  • the promoter used to control transcription in vivo may be any promoter used for any DNA-dependent RNA polymerase.
  • Specific examples of DNA-dependent RNA polymerases are T7, T3, and SP6RNA polymerases.
  • DNA templates for in vitro RNA transcription can be obtained by cloning a nucleic acid (particularly a cDNA) corresponding to a corresponding RNA to be transcribed in vitro and introducing it into a suitable vector for in vitro transcription (e.g., introducing into the plasmid DNA) .
  • the DNA template is linearized with a suitable restriction enzyme and subsequently transcribed in vitro.
  • cDNA can be obtained by reverse transcription or chemical synthesis of mRNA.
  • DNA templates for RNA synthesis in vitro can also be obtained by gene synthesis. (Fotin-Mleczek Mariola et al., CN108064176A)
  • RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotide monomers. These nucleotides are typically monomers of adenosine monophosphate (AMP) , uridine monophosphate (UMP) , guanosine monophosphate (GMP) and cytidine monophosphate (CMP) , or analogues thereof, which are linked to each other along a so-called skeleton. The skeleton is formed by a phosphodiester bond between the sugar (i.e., ribose) of the first monomer and the phosphate moiety of the second adjacent monomer.
  • AMP adenosine monophosphate
  • UMP uridine monophosphate
  • GMP guanosine monophosphate
  • CMP cytidine monophosphate
  • RNA sequence The specific order of monomers, i.e., the order of bases attached to the carbohydrate/phosphate skeleton, is called an RNA sequence.
  • RNA can be obtained by transcription of DNA sequence (for example, in cells) . In eukaryotic cells, transcription typically takes place in the nucleus or mitochondria. In vivo, DNA transcription usually produces so-called premature RNA (also known as pre-mRNA, precursor mRNA or heterologous nuclear RNA) , which must be processed into so-called messenger RNA (usually abbreviated as mRNA) .
  • mRNA messenger RNA
  • RNA maturation Mature messenger RNA usually provides nucleotide sequences that can be translated into amino acid sequences of specific peptides or proteins.
  • the mature mRNA includes a 5′-cap, optional 5′UTR, an open reading frame, optional 3′UTR, and a poly (A) tail (Fotin-Mleczek Mariola et al., CN108064176A) .
  • RNA further includes any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA) , antisense RNA (asRNA) , circular RNA (circRNA) , ribozyme, aptamer, riboswitch, immunostimulating/immunostimulatory RNA, transfer RNA (tRNA) , ribosomal RNA (rRNA) , small nucleolar RNA (snRNA) , small nucleolar RNA (snoRNA) , micro RNA (miRNA) , and Piwi interacting RNA (piRNA) (Fotin-Mleczek Mariola et al.
  • siRNA small interfering RNA
  • asRNA antisense RNA
  • circRNA circular RNA
  • ribozyme aptamer
  • the term “chemically modified” refers to the modification of A, G, U or C ribonucleotides. In general, these terms are not intended to refer to the modifications of ribonucleotide at the naturally occurring 5′end mRNA cap portion. Modifications may be various modifications.
  • the coding region, flanking region and/or terminal region may include one, two or more (optionally different) nucleoside or nucleotide modifications.
  • the modified polynucleotide introduced into the cell may exhibit reduced degradation in the cell as compared to the unmodified polynucleotide.
  • amino acid refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxyl group of -CO 2 H or a sulfo group of -SO 3 H) , wherein an amino acid is linked to a parent molecular group by the side chain, the amino group, or the acid group (e.g., a side chain) .
  • amino acids are linked to parent molecular groups through carbonyl groups, wherein side chains or amino groups are linked to the carbonyl groups.
  • Exemplary side chains include optionally substituted alkyl, aryl, heterocyclic, alkaryl, alkylheterocyclic, aminoalkyl, carbamoylalkyl, and carboxyalkyl.
  • Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolidine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine and valine.
  • the amino acid group may optionally be 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 groups, as defined herein (e.g., unsubstituted amino groups) .
  • Delivery refers to the act or manner of delivering a compound, substance, entity, part, cargo or payload.
  • delivery agent refers to any substance that at least partially promotes in vivo delivery of polynucleotides to target cells.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) generation of an RNA template from a DNA sequence (e.g., by transcription) ; (2) processing of RNA transcripts (e.g., by cutting, editing, 5′ cap formation and/or 3′ terminal processing) ; (3) translation of RNA into peptides or proteins; and (4) post-translational modification ofpeptides or proteins.
  • formulation includes at least one polynucleotide and a delivery agent.
  • 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 in an organism (e.g., an animal, plant, or microorganism) .
  • in vivo refers to events occurring within an organism (e.g., an animal, plant, or microorganism, or its cells or tissues) .
  • Modified refers to a changed state or structure of a molecule according to the present invention. Molecules can be modified by many methods, including chemical, structural and functional modifications.
  • the mRNA molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides, for example, when they relate to natural ribonucleotides A, U, G and C. Atypical nucleotides such as cap structures are not considered “modified” , although they differ from the chemical structures of A, C, G, and U ribonucleotides.
  • Naturally occurring means existing in nature without artificial assistance.
  • pharmaceutically acceptable is used herein to refer to compounds, materials, compositions and/or dosage forms that, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • the term “preventing” refers to partial or complete delay in the onset of an infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, features or clinical manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, features or manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the progression of an infection, particular disease, disorder and/or condition; and/or a reduction of the risk of pathology associated with an infection, disease, disorder and/or condition.
  • Treating refers to partially or completely alleviating, improving, ameliorating and lightening a particular infection, disease, disorder and/or condition; partially or completely delaying the onset of a particular infection, disease, disorder and/or condition; partially or completely inhibiting the progression of a particular infection, diseases, condition and/or condition; partially or completely reducing the severity of a particular infection, disease, disorder and/or condition; and/or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder and/or condition.
  • “treating” cancer may refer to inhibiting the survival, growth and/or spread of tumors.
  • the treating may be administered to subjects who show no signs of the disease, disorder and/or condition and/or subjects who show only early signs of the disease, disorder and/or condition.
  • the reagents used in the present invention are commercially available or can be prepared by the methods described in the present invention.
  • Table 1 illustrates various peptide sequences that may be used to practice the present invention, but it will 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 explicitly written sequences. On the contrary, it will be readily apparent to such persons that based on the teachings set forth above with respect to the peptides described in the general formula, there may be a large number of peptides potentially suitable for practicing the present invention as set forth herein. Furthermore, it is well within the knowledge of those skilled in the art to determine whether a given peptide sequence falls within the scope of the present invention without the need for improper experimentations using standard techniques in the art.
  • variants of the peptide sequences appearing in Table 1 are also within the scope of the present invention, as long as such variants satisfy the structural and functional characteristics as set forth above.
  • Variants of the peptide sequence appearing in Table 1 or of any other candidate peptide not explicitly described in Table 1 but satisfying the structural and functional specific requirements as set forth above may include deletion, insertion, substitution using naturally occurring or non-protein amino acids.
  • Example I preparation method of the peptide of the present invention
  • the non-naturally occurring peptides of the present invention are produced by any previously known peptide synthesis methods 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.
  • the solid phase synthesis method (Marrifield, Journal of the American Chemical Society (J. Am. Chem. Soc. ) , 85, 2149-2154, 1963) may be labeled as only one example of such a peptide synthesis method.
  • peptides can be produced simply and in a relatively short period of time using automated universal peptide synthesizers based on those principles.
  • peptides may be produced using well-known recombinant protein production techniques which are widely known to those skilled in the art.
  • Resin swelling selecting Fmoc-Arg (pbf) -2-Chlorotrityl Resin (molar substitution coefficient 0.317 mmol/g) as a starting resin and adding into a 50ml reaction column, adding DCM (dichloromethane) for soaking, and draining to complete the swelling of the resin.
  • Ninhydrin method is used to monitor the reaction process.
  • step (1) weighing the corresponding amount of TBTU and protective amino acids in a beaker, adding DMF to dissolve; then adding the reaction solution into the resin deprotected in step (1) , then adding DIEA, introducing N 2 and bubbling for about 90 minutes, and performing the detection with the ninhydrin reaction. After finishing the reaction, the solvent is removed and the resin is washed with DMF for 3 times. Then, DMF solution containing 20% (g/100ml) piperidine is added into the resin, N 2 is introduced to continue bubbling for 30 minutes, then the solvent is removed, and the resin is washed with DMF for 6 times, thus finishing the coupling of amino acids.
  • DMF solution containing 20% (g/100ml) piperidine is added into the resin, N 2 is introduced to continue bubbling for 30 minutes, then the solvent is removed, and the resin is washed with DMF for 6 times, thus finishing the coupling of amino acids.
  • Condensation method TBTU + DIEA
  • condensation agent TBTU: 0.64 g
  • DIEA 0.7 ml.
  • the peptide complex nanoparticle of the present invention can be prepared by referring to the nanoparticle preparation method of patents CN111249476A, CN111281981B, CN111281982A, CN111285845B, CN111588637A or an application numbered 202110713076.9.
  • Preparation method I dissolving the peptide in the nuclease-free ultrapure water to obtain a solution of 1 mg/ml, then mixing the peptide and mRNA according to the mass ratio in Table 3 and stirring for 15 minutes, and then standing to obtain the peptide complex nanoparticles.
  • the specific prescriptions are shown in Table 3:
  • Preparation method II according to the prescription design in Table 4, taking PEG derivative auxiliary materials 304, and/or 90R4, and/or 17R4, and/or T90R4R, and/or 704, and/or L64 from a refrigerator at -20°C, balancing to 25 °C, weighing at 25 °C, dissolving in the nuclease-free ultrapure water in a nuclease-free 15 ml centrifuge tube to make its concentration of 100 mg/ml, and fully vortexing with a vortexer for 5 minutes to obtain a stock solution A.
  • Preparation method III weighing 5.0 mg of DMG-PEG and 1 ml of chromatographic pure ethanol for dissolution, weighing 10.3 mg of DSPC (distearoyl phosphatidylcholine) and adding 1 ml of chromatographic pure ethanol for dissolution; and weighing 19.4 mg of cholesterol and adding 2ml of chromatographic pure ethanol for dissolution.
  • DSPC disearoyl phosphatidylcholine
  • Preparation method IV weighing 10 mg of a corresponding auxiliary material, such as DMG-PEG, PEG-CLS or PEG-DSPE in Table 6, dissolving the material by adding the nuclease-free ultrapure water to make its concentration of 1 mg/ml, so as to obtain the stock solution A. Dissolving the peptide in the nuclease-free ultrapure water to obtain a solution of 1 mg/ml, so as to obtain a stock solution B.
  • a corresponding auxiliary material such as DMG-PEG, PEG-CLS or PEG-DSPE in Table 6
  • the mixed solution and mRNA are respectively mixed for 10 minutes, 15min, 30min or 60min, and the peptide complex nanoparticles are obtained by fully vortexing with a vortexer for 20 minutes.
  • the specific prescriptions are shown in Table 6:
  • the prepared aqueous solution of the peptide complex nanoparticles is mixed with a lyophilized agent, and then lyophilized by a lyophilizer (Christ Alpha LD plus, Germany) to prepare a lyophilized agent, which could be trehalose or sucrose, and stored in a refrigerator at 4°C for later use.
  • a lyophilizer Christ Alpha LD plus, Germany
  • Example III characterization of the peptide complex nanoparticles of the present invention
  • Nanoparticle morphology EGFP-mRNA is used as model mRNA, the peptide complex nanoparticle is prepared according to the preparation method I, or the preparation method II, or the preparation method III, or the preparation method IV and the corresponding prescriptions in Example II, and the nanoparticle morphology of the representative peptide complex nanoparticle aqueous solutions of the present invention is tested by using a transmission electron microscope (model FEI Talos F200X) .
  • the copper grid without any dyeing is immersed in the freshly prepared aqueous solution of peptide complex nanoparticles, and naturally dried at 25 °C to prepare samples, which are obtained by testing.
  • FIG. 1 the results show that the peptide complex nanoparticles of the present invention have good dispersibility, present regular or irregular spherical structure, and the particle size ranges from 60 nm to 120 nm.
  • the results are shown in FIG. 1.
  • Particle size and potential EGFP-mRNA is used as model mRNA, the peptide complex nanoparticles are prepared according to the preparation methods described in Example II, and the dynamic light scattering particle size, Zeta potential and polydispersity (PDI) of the peptide complex nanoparticles are measured by Malvern Zetasizer Nano ZSE at 25 °C. The results are shown in Tables 3 to 6. The results show that the peptide complex nanoparticles have a size ranges from 56 nm to 273 nm, with good dispersibility, and the surface charge of the nanoparticles is between -15 mV and 5 mV.
  • Encapsulation rate FLuc-mRNA is used as model mRNA, the peptide complex nanoparticles are prepared according to the preparation methods described in Example II, Quant-iT RiboGreen RNA detection kit (available from ThermoFische Company) is used to determine the mRNA encapsulation rate of each prescription. For the specific method, referring to the instruction of the kit.
  • Example IV agarose gel electrophoresis for detection of ability of peptides to compress mRNA
  • agarose gel Preparing the agarose gel with a mass to volume ratio of 1% (agarose 0.4 g: 1 ⁇ TAE buffer 40 ml) , microwaving twice to melt it fully, adding 4 ⁇ l SyBR Safe DNA Gel Stain dye (Lot No. 1771519, Invitrogen, USA) to the agarose at a ratio of 1 ⁇ 10000, mixing well and pouring into the corresponding gel tank (15-well slot) and cooling for 20 minutes before use.
  • SyBR Safe DNA Gel Stain dye Lot No. 1771519, Invitrogen, USA
  • EGFP-mRNA is used as model mRNA, adding 1 ⁇ l mRNA solution with a concentration of 100 ng/ ⁇ l (i.e., 100 ng) , then adding 9 ⁇ l nuclease-free ultrapure water to make up the volume of the system to 10 ⁇ l, and finally adding 2 ⁇ l of 6*loading buffer to mix evenly.
  • Sample set configuration method according to different mass ratios of peptide to mRNA, 1 ⁇ l of mRNA solution with a concentration of 100 ng/ul (i.e., 100ng) is added into peptide solution (1 ⁇ g/ ⁇ l) and mixed evenly, then nuclease- free ultrapure water is added to make up the volume of the system to 10 ⁇ l, after mixing for 10 minutes, 2 ⁇ l of 6*loading buffer is added to each sample for uniform mixing. After the samples are mixed, 12 ⁇ l system is added to each well, and the gel is run for 25 minutes by 80V voltage electrophoresis instrument, and observed by gel imager. The experimental results are shown in FIG. 2.
  • the minimum mass ratio (peptide: mRNA) of each peptide to ensure that mRNA is fully compressed is: 4 for Seq. 05, 4 for Seq. 12, 2 for Seq. 46, 2 for Seq. 47, 16 for Seq. 49 and 4 for Seq. 53.
  • Example V in vitro cell transfecfion experiment and cytotoxicity investigation of peptide complex nanoparticles
  • FLuc-mRNA is used as model mRNA
  • DC2.4 cell suspension in logarithmic growth phase is aliquoted into 96-well plate at the density of 4 ⁇ 10 4 cells per well, and then put into 37°C, 5%CO2 incubator for static culture.
  • FLuc-mRNA with a concentration of 1 ⁇ g/ ⁇ l is diluted to 0.1 ⁇ g/ ⁇ l in nuclease-free ultrapure water, and take FLuc-mRNA to prepare peptide complex nanoparticles according to the preparation method of different prescriptions described in Example 2, and then respectively dilute them with nuclease-free ultrapure water to 88 ⁇ l of peptide nanoparticle composition containing 10ng/ ⁇ l FLuc-mRNA liquidpeptide, after standing for 10 minutes, the mixture is added to 96-well plate containing 180 ⁇ l opti-MEM culture medium per well at a volume of 20 ⁇ l per well, and each sample is repeated for 4 wells.
  • the culture solution in 96-well plate is replaced with complete culture medium.
  • the complete culture medium is aspirated and washed with PBS, 100 ⁇ l D-Luciferin working solution (working concentration: 250 ⁇ g/ml) is added to each 96-well plate, and then cultured in 37 °C incubator for 5 minutes, the fluorescence expression intensity of FLuc-mRNA is measured by Omega-Fluostar microplate reader. The results are shown in FIG. 3.
  • Cytotoxicity experiment DC2.4 cell suspension in logarithmic growth phase is aliquoted into 96-well plate at the density of 4 ⁇ 10 4 cells per well, and then put into 37 °C, 5%CO 2 incubator for static culture. After 24 hours, FLuc-mRNA with a concentration of 1 ⁇ g/ ⁇ l is diluted to 0.1 ⁇ g/ ⁇ l in nuclease-free ultrapure water, and take FLuc-mRNA to prepare peptide complex nanoparticles according to the preparation method of different prescriptions described in Example 2, and then respectively dilute them with nuclease-free ultrapure water to 88 ⁇ l of peptide nanoparticle composition containing 10ng/ ⁇ l FLue-mRNA liquidpeptide, after standing for l0 minutes, it is added to 96-well plate containing 180 ⁇ l opti-MEM per well at a volume of 20 ⁇ l per well, and each sample is repeated for 4 wells.
  • the culture solution in 96-well plate is replaced with complete culture medium.
  • the complete culture medium is aspirated and washed with PBS for three times, the cell pores without prescription are used as negative control, and the cell-free CCK-8 medium pores are used as blank control, 90 ⁇ l serum-free culture medium and l0 ⁇ l CCK-8 solution are added to each well, and the incubation is continued for 2 hours in the incubator.
  • the absorbance at 450 nm is measured by Omega-FLuostar microplate reader. Calculation formula of cell viability:
  • a (adding medicine) the absorbance of each well added with DC2.4 cells, prescription solution and CCK-8 solution
  • Cell viability cell proliferative activity or cytotoxic activity.
  • Luc-pDNA is used as model mRNA, DC2.4 cell suspension in logarithmic growth phase is aliquoted into 96-well plate at the density of 4 ⁇ 10 4 cells per well, and then put into 37°C, 5%CO 2 incubator for static culture. After 24 hours, Luc-pDNA with a concentration of 1 ⁇ g/ ⁇ l is diluted to 0.1 ⁇ g/ ⁇ l with the nuclease-free ultrapure water.
  • Luc-pDNA take Luc-pDNA to prepare peptide complex nanoparticles according to the preparation method of different prescriptions described in Example 2, and then respectively dilute them with nuclease-free ultrapure water to 88 ⁇ l of peptide nanoparticle composition containing 15ng/ ⁇ l Luc-pDNA liquid, after standing for 30 minutes, the mixture is added to 96-well plate containing 180 ⁇ l opti-MEM culture medium per well at a volume of 20 ⁇ l per well, and each sample is repeated for 4 wells. After 4 hours of administration, the culture solution in 96-well plate is replaced with complete culture medium.
  • the complete culture medium is aspirated, and 100 ⁇ l D-Luciferin solution with a working concentration of 250 ⁇ g/ml is added into each 96-well plate, and then cultured in incubator at 37 °C for 5 minutes, finally, the fluorescence expression intensity of Luc-pDNA is tested by imaging with an Omega-FLuostar microplate reader, the test is repeated every 24 hours, the medium containing D-Luciferin is aspirated after each test, fresh complete culture medium is added to continue culturing for 24 hours, and then D-Luciferin is added for testing, repeated for 3 days. Results are shown in FIG. 5, the abscissa represents different prescriptions, and the ordinate shows a relative fluorescence intensity expressed by Luc-pDNA with the same dose after transfection for 24h, 48h and 72h. The results are shown in FIG. 5.
  • the peptide complex nanoparticles encapsulated with Luc-pDNA shows good expression at cellular level, with the highest expression on the second day and decreasing from the third day, wherein Rp.01, Rp. 27 and Rp. 34 are superior to other prescriptions.
  • Example VI detection of in vivo transfection of peptide complex nanoparticles in mice by small animal fluorescence imaging
  • the blank control group is represented by NC, and 50 ⁇ l PBS buffer is injected intramuscularly with the insulin needle.
  • a proper amount of D-Luciferin is diluted with PBS to prepare a solution with a concentration of 25 mg/ml, keeping away light and spare, each mouse is intraperitoneally injected with 125 ⁇ l substrate, and the mice are placed in a small animal anesthesia box, and a vent valve is opened to release isoflurane to anesthetize the mice.
  • Example VII evaluation of humoral immune effect of peptide complex nanoparticles in mice
  • Novel coronavirus S-mRNA is used as model mRNA, which is provided by Shanghai Hongene Biotech Corporation.
  • the nucleotide sequence of the novel coronavirus S-mRNA (cap1 structure, N1-me-pseudo U Modified) is shown in the sequence table for S-mRNA.
  • S-mRNA stock solution The specific information of S-mRNA stock solution is as follows:
  • Step 1 first immunization in mice: on day 0, female BALB/c mice at 5-6 weeks are divided into 9 groups (5 mice per group) and injected intramuscularly with 75 ⁇ l PBS (blank control) , 5 ⁇ g combination of naked S-mRNA and 5 ⁇ g S protein (positive control) and 75 ⁇ l peptide complex nanoparticle recipe groups Rp. 21, Rp. 25, Rp. 27, Rp. 41, Rp. 01, RP. 08 encapsulated with 5 ⁇ g S-mRNA.
  • Step 2 first serum collection: on the 28th day, blood is collected from the outer canthus of mice.
  • the serum is solidified at 4 °C for 1 hour, then centrifuged at 5000 ⁇ g at 4°C for 5 minutes, removing the supernatant, and then centrifuged at 10000 ⁇ g at 4°C for 5 minutes, removing the supernatant, aliquoted by adding into eight consecutive PCR tubes, and frozen at -20°C for later use.
  • Step 3 second immunization in mice: on the 28th day, after blood collection from the outer canthus, the mice is injected intramuscularly with 75 ⁇ l PBS (blank control) , 5 ⁇ g combination of naked S-mRNA and 5 ⁇ g S protein (positive control) and 75 ⁇ l peptide complex nanoparticle recipe groups Rp. 21, Rp. 25, Rp. 27, Rp. 41, Rp. 01, RP. 08 encapsulated with 5 ⁇ g S-mRNA. Repeating the first immunization process.
  • Step 4 second serum collection: 21 days after the second immunization, blood is collected from the outer canthus of mice.
  • the serum is solidified at 4 °C for 1 hour, then centrifuged at 5000 ⁇ g (5000 times gravity acceleration) at 4 °C for 5 minutes, removing the supernatant, and then centrifuged at 10000 ⁇ g at 4°C for 5 minutes, removing the supernatant, aliquoted by adding into eight consecutive PCR tubes, and frozen at -20°C for later use.
  • Step 5 detecting serum IgG content by ELISA: the S protein is diluted in PBS and the ELISAplate is coated with 100 ⁇ l dilution (containing 1 ⁇ g S protein) per well for 6 hours at 4°C. The liquid in the plate is discarded, 200 ⁇ l PBST is added to each well to wash the plate for 3 times, and then 200 ⁇ l PBS blocking solution containing 5%BSA is added into each well to seal with shaking table at 25°C for 2 hours. The blocking solution is discarded, the plate is washed once with 200 ⁇ l PBST per well, then 100 ⁇ l serum diluted 200 times with PBS is added, and incubated in the shaking table at 25 °C for 2 hours.
  • the serum is discarded, the plate is washed 3 times with 200 ⁇ l PBST per well, and then 100 ⁇ l antibody (diluted with PBS at a ratio of 1 ⁇ 1000) is added to each well, and incubated in the shaking table at 25 °C for 1 hour. After the antibody is discarded, the plate is washed with 200 ⁇ l PBST for three times, and then 50 ⁇ l TMB substrate is added to avoid light. After the positive control well turned dark blue or the reaction lasted for 10 minutes, 5 ⁇ l 2M sulfuric acid is added to stop the reaction. The optical density at the wavelengths of 450 nm and 630 nm wavelengths is detected by ELIASA, and the difference of OD values is calculated to reflect the level of anti-S protein IgG in the serum. The results are shown in FIG. 7.
  • Step 6 serum IgG titer detected by ELISA: the S protein is diluted in PBS and the ELISA plate is coated with 100 ⁇ l dilution (containing 1 ⁇ g S protein) per well for 6 hours at 4°C. The liquid in the plate is discarded, 200 ⁇ l PBST is added to each well to wash the plate for 1 times, and then 200 ⁇ l PBS blocking solution containing 5%BSA is added into each well to seal with the shaking table at 25 °C for 2 hours.
  • the blocking solution is discarded, the plate is washed three times with 200 ⁇ l PBST per well, then the serum diluted 50, 250, 1250, 6250, 31250, 156250, 781250 and 3906250 times with PBS at a ratio of 1 ⁇ 3 are added, and incubated in the shaking table at 25 °C for 2 hours.
  • the serum is discarded, the plate is washed 3 times with 200 ⁇ l PBST per well, and then 100 ⁇ l antibody (diluted with PBS at a ratio of 1 ⁇ 1000) is added to each well, and incubated in the shaking table at 25 °C for 1 hour.
  • the plate is washed with 200 ⁇ l PBST for three times, and then 50 ⁇ l TMB substrate is added to avoid light. After the positive control well turned dark blue or the reaction lasted for 10 minutes, 5 ⁇ l 2M sulfuric acid is added to stop the reaction. The optical density at 450 nm and 630 nm is detected by ELIASA. The results are shown in FIG. 8.
  • the present invention takes 2 times of the average OD value of PBS group as the baseline, and the OD value of Rp. 08 group is still 2 times higher than the baseline when diluted to 3906250 times, suggesting that the recipe group Rp.08 has strong serum transformation efficiency and humoral immune activation function.
  • the gene transfection kit is a versatile transfection reagent that can be composed of any of the prescriptions of the present invention and provides efficient transfection in a variety of adherent and suspension cell lines. It is suitable for all common cell lines and many cell lines that are difficult to transfect, and can be used in medium containing or not containing serum.
  • the kit of the present invention is used to transfect mammalian cells with a 96-well cell culture plate. The specific steps are as follows:
  • the cells are incubated in a CO 2 incubator and the original medium is replaced with complete medium after 4 hours, the incubation is continued in the CO 2 incubator for 12 hours to 72 hours, finally an expression amount of the nucleic acids is detected.

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Abstract

A peptide, a peptide complex nanoparticle, a nucleic acid vaccine and applications thereof are provided. A peptide compound is also provided, which has a following structure of general formula (I): (Xaa)x-Arg-Val-Gln-Pro-Thr-Glu-Ser-Ile-Val-Arg- (Yaa)y (General Formula (I)), wherein: x is an integer from 1 to 25, y is an integer from 0 to 10, (Yaa)y is a peptide segment consisting of any amino acid, and (Yaa)y is a peptide segment consisting of any amino acid.

Description

PEPTIDE AND PEPTIDE COMPLEX NANOPARTICLE, NUCLEIC ACID VACCINE AND APPLICATION THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Chinese Provisional Application No. 202010916860.5, filed September 03, which is incorporated by reference herein.
Technical Field:
The present invention relates to the field of drug delivery, and particularly to a peptide and a peptide complex nanoparticle, a nucleic acid vaccine and applications thereof.
Background
Gene transfection is a technology for transferring or transporting nucleic acids with biological functions into cells and making nucleic acids maintain the biological functions thereof in the cells. A gene vector is a tool for introducing exogenous therapeutic genes into biological cells. Nucleic acid vaccine is a new type of vaccines that have been developed in recent years. As a new response option, the nucleic acid vaccine is to introduce nucleic acid encoding antigen protein into cells and synthesize the protein through a cell expression system, so as to induce specific immune response. Although most cells can spontaneously take up nucleic acids, the efficiency is very low and the cells are saturated at low  doses. In addition, the presence of a large number of RNA enzymes (RNases) in nature makes RNA very unstable and susceptible to degradation both in vitro and in vivo. Therefore, suitable agents are needed to protect nucleic acids from extracellular RNase-mediated degradation and to promote the nucleic acids entry into cells. In the development of both preventive and therapeutic nucleic acid vaccines, delivery of specific sequences of nucleic acids to dendritic cells (DCs) for safe, efficient and sufficient expression is essential for vaccine efficacy.
Cell penetrating peptides (CPPs) are a class ofpeptides that can directly cross cell membranes without causing damage to the cell membrane in a non-receptor-dependent, non-classical endocytosis manner, and are generally less than 30 amino acids in length and rich in basic amino acids, usually with positively charged amino acid sequences, such as the human immunodeficiency virus-1 transcription activator TAT (HIV-1 TAT) (Vives et al., J. Biol. Chem. 1997; 272, 16010) . The common properties of CPPs are: net positive or electroneutral charge, hydrophilicity and hydrophobicity (amphipathy) ; high efficiency of membrane penetration delivery; low cytotoxicity; no cell type restriction; and the ability to introduce different bioactive substances into cells by chemical binding or gene fusion, making them potentially versatile targeted drug carriers. The specific transmembrane mechanisms vary among CPPs, and particular amino acid sequences have been found to bind to mRNA and to interfere with and reduce cell membrane stability, thereby carrying bioactive substances across the cell membrane, such as the arginine-alanine-leucine-alanine residue (RALA)  sequence (Pardi et al. Curt Opin in Immunol. 2020, 65: 14 -20) . Compared to other delivery modalities, there is still a lack of reported research on CPPs in the field of mRNA delivery or vaccine development.
The advantage of preparing peptides linked by amide bonds (peptide bonds) into peptide complex nanoparticles is their ability to degrade into amino acids in vivo while ensuring high transfection efficiency and low cytotoxicity, however, there is no peptide complex nanoparticle delivery system that enables mRNA-based gene drugs to be marketed.
The present invention addresses the shortcomings of existing delivery systems, synthesizes non-naturally occurring peptides, and prepares peptide complex nanoparticles to provide improved gene vector for mRNA delivery and nano-delivery solutions suitable for animal vaccine or human mRNA drug development.
Summary
Brief Description of Invention
To solve the above problems, in a first aspect, the present invention provides a peptide compound for nucleic acid drug delivery. In a second aspect, the present invention provides a peptide complex nanoparticle containing the peptide compound. In a third aspect, the present invention provides an application of a peptide complex nanoparticle in nucleic acid delivery in vitro and in vivo. In a fourth aspect, the present invention provides a nucleic acid vaccine containing the  peptide complex nanoparticles. In a fifth aspect, The present invention provides a use of the peptide complex nanoparticles in preparing medicines or kits.
Detailed Description of Invention
In the first aspect, the present invention provides a peptide compound having a following structure of general formula I:
(Xaa)  x-Arg-Val-Gln-Pro-Thr-Glu-Ser-Ile-Val-Arg- (Yaa)  y (General Formula I)
where x is an integer from 1 to 25 and y is an integer from 0 to 10;
(Xaa) x is a peptide segment composed of any amino acid. 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) , x is the number of amino acids, with x being 1 to 20. In some embodiments, (Xaa) x is Arg. In some embodiments, (Xaa)  x is (Xa′a′)  n (Arg)  1-10 (Xa′a′)  n, where Xa′a′ is selected from 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 some embodiments, Xaa consists of (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) , with Trp (W) , Cys (C) , His (H) , and/or Pro (P) either preceding or following (Arg)  1- 10, or interspersed in one or several (Arg)  1-10 in between.
In general formula I, a sequence of Arg (R) , Trp (W) , Cys (C) , Lys (K) , Leu (L) , Phe (F) , Pro (P) or His (H) in amino acid sequences is not limited.
(Yaa) y is a peptide segment consisting of any amino acid; in some  embodiments, Yaa is selected from at least one of Arg (R) , Trp (W) , Phe (F) , or Cys (C) , and y is the number of amino acids, with y being 0 to 10,.
The x can be1, 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.
The y can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
The n can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In some embodiments, an amino acid sequence of the peptide 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 peptide compound is: Seq. 05, Seq. 12, Seq. 46, Seq. 47, Seq. 49 or Seq. 53.
In some embodiments, the general formula (I) is at least 50%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 20%; in some embodiments, the general formula (I) is at least 75%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 50%; in some embodiments, the general formula  (I) is at least 90%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 100%; and
In some embodiments, the general formula (I) is at least 90%similar to any one of RRRRRWCRVQPTESIVR, RRRRRWFCRVQPTESIVR, FCRWCRRVQPTESIVRRCWRCF, FCRWCRRVQPTESIVCWRRRCF, HKRWCRRWCRVQPTESIVRC or WCRRRVQPTESIVRRRWC.
In some embodiments, the peptide of general formula (I) includes 10-35 amino acids, which is characterized in that the peptide improves the delivery of nucleic acid molecules into the cell by at least 10%, in some embodiments, the peptide of general formula (I) includes 10-35 amino acids, which is characterized in that the peptide improves the delivery of nucleic acid molecules into the cell by between about 50%and about 100%; and in some embodiments, the peptide of general formula (I) includes 10-35 amino acids, which is characterized in that the peptide improves the delivery of nucleic acid molecules into the cell by between about 75%and about 500%.
The present invention provides novel non-naturally occurring peptides with functions such as compressing and protecting nucleic acids from degradation and facilitating penetration of nucleic acids through cell membranes, as well as peptide complex nanoparticles containing the peptides, and methods for applying the peptide complex nanoparticles to gene transfection of cells in vivo and in vitro, and methods of applying the peptide complex nanoparticles to vaccine formulations.
In the second aspect, the present invention provides a peptide complex nanoparticle.
A peptide complex nanoparticle, which includes:
a) at least one peptide of the first aspect of the present invention and
b) a nucleic acid.
In some embodiments, a peptide complex nanoparticle, which includes:
a) at least one peptide of the first aspect of the present invention and a composition of at least one auxiliary material; and
b) a nucleic acid.
The nucleic acid may be chemically modified or unmodified DNA, single-stranded or double-stranded DNA, coding or non-coding DNA. In some embodiments, the nucleic acid is selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA primers, DNA probes, immunostimulatory DNA, aptamers, or any combination thereof.
The nucleic acid may 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) , oligonucleotides, viral RNA, replicon RNA, transfer RNA (tRNA) , ribosomal RNA (rRNA) , immunostimulatory RNA (isRNA) , micro RNA, small interfering RNA (siRNA) , small nuclear RNA (snRNA) , small hairpin RNA (shRNA) or  riboswitch, RNA aptamer, RNA decoy, antisense RNA, nuclease, or any combination thereof. In some preferred embodiments, the nucleic acid is a chemically modified messenger RNA (mRNA) .
The nucleic acid sequences of the RNA may include all the nucleic acid sequences listed in patent US9254311B2, also include all the sequences listed in the long sequence appendix of the patent. In some embodiments, the RNA sequences described in the present invention can be obtained by the nucleic acid synthesis method listed in patent US9254311B2 or CN106659803A.
The peptide complex nanoparticles can encapsulate mRNA and allow its efficient introduction into different cell lines in vitro and can be efficiently transfected in vivo. The peptide complex nanoparticle of the present invention can carry mRNA encoding an immunogenic peptide into cells and effectively release the mRNA to express antigens and effectively achieve immunotherapy or immunoprophylaxis.
The present invention provides novel non-naturally occurring peptides with functions such as compressing and protecting nucleic acids from degradation and facilitating penetration of nucleic acids through cell membranes, as well as peptide complex nanoparticles containing the peptides, and methods for applying the peptide complex nanoparticle to gene transfection of cells in vivo and in vitro, and methods of applying the peptide complex nanoparticle to vaccine formulations.
In another aspect, the present invention provides a peptide complex nanoparticle, which includes:
a) at least one peptide of the present invention,
b) a nucleic acid, and
c) an auxiliary material.
The auxiliary material may be selected from one or more of phospholipids, PEG lipids or PEG derivatives.
The lipid may be a naturally occurring or synthetic phospholipid or structural lipid.
The PEG derivative may be poloxamide, poloxamide derivative, poloxamer derivative or PEG lipid.
The poloxamide can be selected from at least one of the following poloxamide: 
Figure PCTCN2021116359-appb-000001
Figure PCTCN2021116359-appb-000002
Figure PCTCN2021116359-appb-000003
or 
Figure PCTCN2021116359-appb-000004
and The poloxamide derivatives are synthesized with reference to patent CN111285845B.
The poloxamer can be selected from at least one of the following poloxamer: 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 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.
The phospholipids can be selected from: 1, 2-distearoyl-sn-glycerol-3-phosphate choline (DSPC) , 1, 2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE) , 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DLPC) , 1, 2-dimyristoyl-sn-glycerol-phosphate choline (DMPC) , 1, 2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC) , 1, 2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC) , 1, 2-bis-undecanoyl-sn-glycerol-phosphate choline (DUPC) , 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) , 1, 2-di-o-octadecenyl-sn-glycerol-3-phosphate choline (18∶0 diethyl PC) , 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycerol-3-phosphate choline (ochemspc) 1-hexadecyl-sn-glycerin-3-phosphate choline (C 16 LysoPC) , 1, 2-dioleoenyl-sn-glycerin-3-phosphate choline, 1, 2-diaarachidonyl-sn-glycerin-3-phosphate choline, 1, 2-bis (docosahexaenoyl) -sn-glycerin-3-phosphate choline, 1, 2-diphytanoyl-sn-glycerin-3-phosphate ethanolamine (me 16.0, PE) , 1, 2-distearoyl-sn-glycerin-3-phosphateethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-diaarachidonicacid-sn-glycerol-3-phosphate ethanolamine, 1, 2-didecahexaenoyl-sn-glycerol-3-phosphate ethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphate racemic- (1- glycerol) sodium salt (DOPG) , sphingomyelin, or lecithin (PC) and mixtures thereof;
the structural lipids may be selected from: cholesterol (Chol) , stersterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and mixtures thereof; and
The PEG lipid may be selected from any of the PEG lipids described in Patent Nos. CN111281981B, CN111315359A, CN111356444A, such as 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-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) , such as mPEG5000-C-CLS (PEG-CLS) , such as mPEG2000-DSPE (PEG-DSPE) .
The 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG) has the following structural formula:
Figure PCTCN2021116359-appb-000005
DMG-PEG.
The mPEG5000-C-CLS (PEG-CLS) has the following structural formula:
Figure PCTCN2021116359-appb-000006
PEG-CLS.
The mPEG2000-DSPE (PEG-DSPE) has the following structural formula:
Figure PCTCN2021116359-appb-000007
PEG-DSPE
A mass ratio of the nucleic acid to the peptide may be less than or equal to about 1∶1. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶1 to about 1∶52. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶48. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶40. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶32. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶24. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶16. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶10. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 toabout 1∶8. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶5. In some embodiments, the mass ratio of the nucleic acid to the peptide is 1∶2  to 1∶4. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶1, 1∶2, 1∶3, 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.
In some embodiments, an amino acid sequence of the peptide is Seq. 05, and the mass ratio of the nucleic acid to the peptide is less than or equal to 1∶4. In some embodiments, the amino acid sequence of the peptide is Seq. 05, and the mass ratio of the nucleic acid to the peptide is about 1∶4 to about 1∶52.
In some embodiments, the amino acid sequence of the peptide is Seq. 12, and the mass ratio of the nucleic acid to the peptide is less than or equal to 1∶4. In some embodiments, the amino acid sequence of the peptide is Seq. 12, and the mass ratio of the nucleic acid to the peptide is about 1∶4 to about 1∶52.
In some embodiments, the amino acid sequence of the peptide is Seq. 46, and the mass ratio of the nucleic acid to the peptide is less than or equal toabout 1∶2. In some embodiments, the amino acid sequence of the peptide is Seq. 46, and the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶52.
In some embodiments, the amino acid sequence of the peptide is Seq. 47, and the mass ratio of the nucleic acid to the peptide is less than or equal to about 1∶2. In some embodiments, the amino acid sequence of the peptide is Seq. 47, and the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶52.
In some embodiments, the amino acid sequence of the peptide is Seq. 49, and  the mass ratio of the nucleic acid to the peptide is less than or equal to about 1∶2. In some embodiments, the amino acid sequence of the peptide is Seq. 49, and the mass ratio of the nucleic acid to the peptide is about 1∶16 to about 1∶52.
In some embodiments, the amino acid sequence of the peptide is Seq. 53, and the mass ratio of the nucleic acid to the peptide is less than or equal to about 1∶4. In some embodiments, the amino acid sequence of the peptide is Seq. 53, and the mass ratio of the nucleic acid to the peptide is about 1∶4 to about 1∶52.
The mass ratio of the nucleic acid to the auxiliary materiale may be less than or equal to about 1∶2. In some embodiments, the mass ratio of the nucleic acid to the auxiliary materiale 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 to about 1∶800. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶500. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶50. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶33. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶10. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 , about 1∶3, about 1∶4, about 1∶5 or about 1∶6. In some embodiments, the mass ratio of the nucleic acid to the auxiliary  material is about 1∶33 to about 1∶400. In some embodiments, the mass ratio of the nucleic acid to the auxiliary material is about 1∶50 to about 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 about 1∶50 to about 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 about 1∶100 , about 1∶200 or about 1∶500. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2, the auxiliary material is a PEG derivative, and the mass ratio of the nucleic acid to the auxiliary material is about 1∶50 to about 1∶800.
In some embodiments, the auxiliary materials are PEG derivatives, phospholipids and structural lipids, and the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to 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 material is about 1∶2 , about 1∶3, about 1∶4, about 1∶5 or about 1∶6. In some embodiments, the mass ratio of the nucleic acid to the peptide is about 1∶2 to about 1∶30, the auxiliary materials are PEG derivatives, phospholipids and structural lipids, , and the mass ratio of the nucleic acid to the auxiliary material is about 1∶2 to about 1∶6.
In some embodiments, the auxiliary materials are PEG derivatives and phospholipids, and the mass ratio of the nucleic acid to the auxiliary material is about 1∶33 to about 1∶400 or about 1∶33 to about 1∶370. In some embodiments, the auxiliary materials are PEG derivatives and phospholipids, the mass ratio of  the nucleic acid to the auxiliary material is about 1∶33 to about 1∶400 or about 1∶33 to about 1∶370, and the mass ratio of the PEG derivative and phospholipid is 32∶1 to 700∶1. In some embodiments, the auxiliary materials are PEG derivatives and phospholipids, the mass ratio of the nucleic acid to the auxiliary material is 1∶2, the mass ratio of the nucleic acid to the auxiliary material is about 1∶33 to about 1∶400 or about 1∶33 to about 1∶370, and the mass ratio of the PEG derivative and phospholipid is about 32∶1 to 700∶1.
In some embodiments, a peptide complex nanoparticle composition, which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 05 and auxiliary materials, the auxiliary materials are
Figure PCTCN2021116359-appb-000008
and lecithin , the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 05, 
Figure PCTCN2021116359-appb-000009
and lecithin is about 1∶2∶322∶1.
In some embodiments, A peptide complex nanoparticle composition, which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 49 and auxiliary materials, the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 49, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol is about 10∶300∶8∶16∶31.
In some embodiments, a peptide complex nanoparticle composition, which comprises: a nucleic acid, the peptide compound with the amino acid sequence of  Seq. 53 and auxiliary materials, the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 53, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol is about about 10∶40∶8∶16∶5.
In some embodiments of the present invention, a peptide complex nanoparticle includes at least one non-naturally occurring peptide and nucleic acid of the present invention.
In some embodiments of the present invention, a peptide complex nanoparticle includes at least one non-naturally occurring peptide, nucleic acid and a least one lipid or PEG derivative of the present invention.
The nucleic acid may be chemically modified or unmodified DNA, single-stranded or double-stranded DNA, coding or non-coding DNA. In some embodiments, the nucleic acid is selected from plasmids, oligodeoxynucleotides, genomic DNA, DNA primers, DNA probes, immunostimulatory DNA, aptamers, or any combination thereof.
The nucleic acid may 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) , oligonucleotides, viral RNA, replicon RNA, transfer RNA (tRNA) , ribosomal  RNA (rRNA) , immunostimulatory RNA (isRNA) , micro RNA, small interfering RNA (siRNA) , small nuclear RNA (snRNA) , small hairpin RNA (shRNA) or riboswitch, RNA aptamer, RNA decoy, antisense RNA, nuclease, or any combination thereof. In some preferred embodiments, the nucleic acid is a chemically modified messenger RNA (mRNA) .
The nucleic acid sequences of the RNA may include all the nucleic acid sequences listed in patent US9254311B2, also include all the sequences listed in the long sequence appendix of the patent. In some embodiments, the RNA sequences described in the present invention can be obtained by the nucleic acid synthesis method listed in patent US9254311B2 or CN106659803A.
In some embodiments, the peptide complex nanoparticle may further include at least one pharmaceutically acceptable excipient.
The peptide complex nanoparticle of the present invention is stable in aqueous solution and can be contacted with human or animal cell tissues after formation, or can be stored for a period of time before contact with the cells or tissues. The peptide complex nanoparticle is stable, and can be stored for 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. It should be understood that the storage period may be between any of these time periods for example between 31 minutes and 1 hour or between 1 hour and 24 hours.
In the third aspect, the present invention provides an application of a peptide complex nanoparticle in nucleic acid delivery in vitro and in vivo.
An application of the peptide complex nanoparticle of the second aspect in nucleic acid delivery in vitro and in vivo is provided.
The peptide complex nanoparticle can encapsulate mRNA and allow its efficient introduction into different cell lines in vitro and can be efficiently transfected in vivo. The peptide complex nanoparticle of the present invention can carry mRNA encoding an immunogenic peptide into cells and effectively release the mRNA to express antigens and effectively achieve immunotherapy or immunoprophylaxis.
The present invention provides novel non-naturally occurring peptides with functions such as compressing and protecting nucleic acids from degradation and facilitating penetration of nucleic acids through cell membranes, as well as peptide complex nanoparticle containing the peptides, and methods for applying the peptide complex nanoparticle to gene transfection of cells in vivo and in vitro, and methods of applying the peptide complex nanoparticle to vaccine formulations.
In the fourth aspect, the present invention provides a nucleic acid vaccine  containing the pepfide complex nanoparticle of the second aspect.
A nucleic acid vaccine is provided, the nucleic acid vaccine includes the peptide complex nanoparticle of the second aspect.
The peptide complex nanoparticle may include at least one RNA.
The nucleic acid vaccine can be used for treating or preventing diseases.
The RNA includes at least coding RNA.
The encoding RNA may include RNA capable of encoding at least one coding region of at least one therapeutic protein, therapeutic peptide, immunogenic protein or immunogenic peptide. In some embodiments, the coding RNA is mRNA.
The present invention provides a RNA vaccine with RNA (such asmessenger RNA (mRNA) ) as the core and peptide complex nanoparticle as the delivery agent, which can safely induce the naturally occurring specific immune system of the body to produce almost any protein of interest or its fragment, including infectious pathogen vaccines such as bacteria and viruses and tumor vaccines. In some embodiments, RNA is modified. The nucleic acid vaccine disclosed by the invention can be used for inducing immune responses against infectious pathogens or cancers, including cellular immune responses and humoral immune responses, without the risk of insertion mutagenesis that may result. Depending on the pathogen of infectious diseases and the incidence of cancer, nucleic acid vaccines with peptide complex nanoparticle as delivery agents can be used in  various disease types. The nucleic acid vaccine can be used for preventing and/or treating infectious pathogens or cancers of various metastatic stages or degrees.
In some embodiments of the present invention, a nucleic acid vaccine is provided, the nucleic acid vaccine includes the peptide complex nanoparticle of the second aspect; the peptide complex nanoparticle includes at least one RNA; the RNA is messenger RNA (mRNA) ; the messenger RNA (mRNA) can safely direct the body′scellular mechanisms to produce almost any protein of interest, from natural proteins to antibodies and other completely novel protein constructs that can be therapeutically active inside and outside the cell.
The nucleic acid vaccine is available in a variety of contexts depending on 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 advantage of the nucleic acid vaccine is that it produces a much larger antibody titer compared to commercially available antiviral treatment and reacts earlier. Although it is not desirable to be bound by theory, it is believed that RNA vaccines, like mRNA polynucleotides, are better designed to produce appropriate protein conformation by translocation when the RNA vaccine assigns a natural cellular mechanism. Unlike conventional vaccines, which are manufactured in vitro and can trigger adverse cellular responses, the nucleic acid vaccine provides a template for the cellular system to express protein antigens in a more natural manner.
In some embodiments of the present invention, a nucleic acid vaccine is provided, the nucleic acid vaccine includes the peptide complex nanoparticle of the second aspect; the peptide complex nanoparticle includes 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 whose nucleotide sequence encodes the S spike protein of novel coronavirus SARS-CoV-2.
In some embodiments of the present invention, a nucleic acid vaccine is provided, the nucleic acid vaccine includes the peptide complex nanoparticle of the second aspect; the peptide complex nanoparticle contains artificially synthesized pathogen antigenic peptides. In some embodiments, the antigenic peptide is fused with other peptides that enhance transfection and delivery efficiency and/or enhance immune response.
The dosage form of the nucleic acid vaccine can be injection, tablet, inhalation formulation, suppository, eye drop or suspension, etc..
The nucleic acid vaccine of the present invention can be administered by any route that produces a therapeutically effective result. The routes include, but are not limited to, intradermal, subcutaneous, intraperitoneal, oral, intramuscular, intranasal, intraocular, upper respiratory, intravenous, vaginal, rectal administration. In some embodiments, the mRNA vaccine of the present invention is administered by injection.
In the fifth aspect, the present invention provides a use of the peptide complex nanoparticle of the second aspect in preparing medicines or kits.
A use of the peptide complex nanoparticle of the second aspect in preparing medicines or kits is provided.
In some embodiments, a use of the peptide complex nanoparticle of the second aspect preparing medicines for the prevention, treatment and/or amelioration of a disease selected from the group consisting of: cancer or rumor diseases, infectious diseases, autoimmune diseases, allergic reaction or allergic disease, monogenic hereditary diseases, or a general genetic disease, diseases with genetic background and typically caused by identified genetic defects and inherited according to Mendelian law, cardiovascular diseases, neuronal diseases, respiratory diseases, digestive diseases, skin diseases, musculoskeletal diseases, connective tissue diseases, rumors, immunodeficiency, endocrine, nutritional and metabolic diseases, eye diseases and ear diseases.
The infectious diseases may include viral infectious diseases, bacterial infectious diseases or protozoological infectious diseases.
Brief Description of the Drawings
FIG. 1 shows a transmission electron microscopic diagram of the peptide complex nanoparticle of Example III; where A represents recipe Rp. 05, B represents recipe Rp. 28, C represents recipe Rp. 43, and the white scale is 200 nm.
FIG. 2 shows agarose gel electrophoresis results of the peptide complex  nanoparticles of Example IV; where mRNA refers to mRNA positive control group, and 1, 2, 4, 8, 16, 32 and 64 refer to the mass ratio of the peptide to mRNA is 1∶1, 2∶1, 4∶1, 8∶1, 16∶1, 32∶1 and 64∶1; and the minimum mass ratio of each peptide to ensure that mRNA is fully compressed is: 4 for Seq. 05, 4 for Seq. 12, 2 for Seq. 46, 2 for Seq. 47, 16 for Seq. 49 and 4 for Seq. 53.
FIG. 3 shows a transfection of FLuc-mRNA peptide complex nanoparticles in DC2.4 cells in Example V; where the abscissa represents peptide nanoparticle compositions of different prescriptions, and the ordinate represents a relative fluorescence intensity expressed after transfection of the peptide nanoparticle compositions containing the same dose of FLuc-mRNA for 24 hours.
FIG. 4 shows a survival rate of DC2.4 cells treated with different prescriptions in Example V; where the abscissa represents different prescriptions of peptide complex nanoparticles, and the ordinate represents cell activity, the higher the cell activity, the smaller the cytotoxicity.
FIG. 5 shows a transfection of Luc-pDNA peptide complex nanoparticles in DC2.4 cells in Example V; the abscissa represents different prescriptions, and the ordinate shows a relative fluorescence intensity expressed by DC2.4 cells at 24h, 48h and 72h after transfection of Luc-pDNA with the same dose.
FIG. 6 shows an expression of luciferase of peptide complex nanoparticles in mice detected by IVIS (In Vivo Imaging Systems) in Example VI.
FIG. 7 shows a serum IgG antibody level of mice immunized with peptide  complex nanoparticles in Embodiment VII; the abscissa represents the 28th and 49th days after the first immunization of different prescriptions, and the ordinate represents the difference of OD value of optical density at two wavelengths, OD value is an index to judge the level of IgG antibody in serum, reflecting the level of anti-S protein IgG in serum.
FIG. 8 shows a serum IgG antibody titer of mice immunized with peptide complex nanoparticles in Embodiment VIII; the abscissa represents different dilution times of serum after 49 days after the first immunization with different prescriptions, and the ordinate represents the difference of OD value (optical density value) at two wavelengths. 2x Baseline (double of background) is used as the cut-off value to distinguish positive and negative results, and the maximum dilution of the OD value higher than this value is the titer.
Definition of Terms
The terms used throughout this description generally have their ordinary meaning in the field to which they belong within the context of the present invention and in the particular circumstances in which each term is used. Certain terms are discussed below or elsewhere in the description to provide practitioners with additional guidance in 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 may be used for any one or more of the terms discussed  herein, whether or not a term is detailed or discussed herein, and without assigning any particular meaning. Synonyms for certain terms may be provided. Recitation of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in this description (including examples of any of the terms discussed herein) is illustrative only and in no way limits the scope or meaning of the present invention or any of the terms exemplified.
The terms "about" as used herein represent an amount close to the stated amount that still performs the desired function or achieves the desired result. For example, the terms "about" may refer to an amount that is within less than 10%of, within less than 5%of, within less than 1%of, within less than 0.1%of, and within less than 0.01%of the stated amount.
The term “peptide” refers to a polymer of amino acid residues (natural or non-natural) that are in many cases linked together by peptide bonds. As used herein, the term refers to proteins, peptides, and peptides of any size, structure, or function. Peptides may be single molecules or may be multimolecular complexes such as dimers, trimers, or tetramers. They can also contain single-chain or multi-chain peptides such as antibodies or insulin, and can be associated or linked. The most common disulfide bonds are found in multi-chain peptides. The term peptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of corresponding naturally occurring amino acids.
The term “protein” refers to a polymer consisting essentially of any of the 20 amino acids. Although “peptide” is usually used to refer to relatively large peptides and “peptide” is usually used to refer to small peptides, the use of these terms in the field overlaps and varies. As used herein, the terms “peptide” , “protein” and “peptide” are sometimes used interchangeably.
The term “hydrophilic” means soluble in water under specific conditions, including readily soluble in water, soluble in water and slightly soluble in water.
The term “hydrophobic” means poorly soluble in water under certain conditions.
As used herein, the term “amino acid” generally refers to naturally occurring or synthetic amino acids, as well as amino acid analogues and amino acid mimetics that act in a similar manner to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those subsequently modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphate serine. Amino acid analogues refer to compounds having the same basic chemical structure as naturally occurring amino acids (i.e., alpha carbon bound to hydrogen, carboxyl, amino and R groups) , such as homoserine, n-leucine, methionine sulfoxide, methionine and methyl sulfonium. Such analogues have a modified R group (e.g., norleucine or norvaline) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds having a  structure different from the general chemical structure of amino acids but function in a similar way to naturally occurring amino acids. The term “amino acid” may refer to amino acids or their derivatives (e.g., amino acid analogues) and their 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 acid, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, N-acetylcysteine.
“Kit” means a transfection, DNA, RNAi or other bioactive (e.g., protein or anion molecule) delivery or protein expression or knockdown kit that includes one or more reagents of the present invention or mixtures thereof. The kit may include one or more non-naturally occurring peptides described herein, or optionally in combination with one or more lipids or PEG derivatives. In some embodiments, that peptide and lipid agent may be provided in the form of a single formulation. In other embodiments, the complex material and peptide may be provided separately, together with instructions instructing the user to combine the reagents in use. Such kits may include carrying devices that are partitioned to hold one or more container devices (e.g., vials, test tubes, etc. ) in a tightly confined manner. Each of these container devices contains a component or mixture of components 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..
The media, methods, kits and compositions of the present invention are suitable for monolayer or suspension culture, transfection and cultivation of cells and for expression of proteins in monolayer or suspension cultured cells. In some embodiments, the media, methods, kits and compositions of the present invention are used for suspension culture, transfection and cultivation of cells, and for expression of protein products in suspension cultured cells.
Immune response: the immune response can typically either 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)
Vaccine: vaccine is typically understood as a prophylactic or therapeutic substance that provides at least one antigen or antigenic function. The antigen or antigen function can stimulate the adaptive immune system of the body to provide an adaptive immune response. (Fotin-Mleczek Mariola et al., CN108064176A) 
mRNA for providing antigen: mRNA for providing antigen can typically be mRNA with at least one open reading frame, which can be translated by cells or organisms provided with the mRNA. The translated product is a peptide or protein which can be used as an antigen, preferably as an immunogen. The product can  also be a fusion protein composed of more than one immunogen, for example, a fusion protein composed of two or more epitopes, peptides or protein, wherein the epitopes, peptides or protein can be connected by a connecting sequence. (Fotin-Mleczek Mariola et al., CN108064176A)
Nucleic acid: the term “nucleic acid” refers to any DNA or RNA molecule and is used synonymously with “polynucleotide” . Wherever a nucleic acid or nucleic acid sequence encoding a particular protein and/or peptide is mentioned herein, the nucleic acid or nucleic acid sequence preferably further includes regulatory and/or other sequences that allow their expression and/or stability in a suitable host (e.g., human) , i.e., transcription and/or translation of the nucleic acid sequence encoding a particular protein or peptide. (Fotin-Mleczek Mariola et al., CN108064176A)
Peptide: Peptides are polymers of amino acid monomers. Usually, 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 present invention, the peptide may for example contain less than 50 monomer units. Longer peptides may also be referred to as peptides and typically have 50 to 600 monomer units more specifically 50 to 300 monomer units. (Fotin-Mleczek Mariola et al., CN108064176A)
Pharmaceutically effective amount: in the context of the present invention, the pharmaceutically effective amount is typically understood as an amount  sufficient to induce an immune response or trigger a desired therapeutic effect. (Fotin-Mleczek Mariola et al., CN108064176A)
Protein: proteins typically consist of one or more peptides and/or peptides that fold into a three-dimensional form and promote a biological function. (Fotin-Mleczek Mariola et al., CN108064176A)
Chemical synthesis of RNA: the chemical synthesis of relatively short segments of an oligonucleotide having a defined chemical structure provides a fast and inexpensive way to obtain a customized oligonucleotide of any desired sequence. Although enzymes only synthesize DNA and RNA in the 5′ to 3′ direction, chemical oligonucleotide synthesis does not have this limitation, although it is most often carried out in the opposite direction (i.e., 3′ to 5′) . At present, this process is carried out in the form of solid phase synthesis using phosphoramidite methods and phosphoramidite structural units derived from protected nucleotides (A, C, G and U) or chemically modified nucleotides. (Fotin-Mleczek Mariola et al., CN108064176A)
In order to obtain the desired oligonucleotide, the structural units are sequentially coupled to the growth oligonucleotide chain in the solid phase in the order required by the product sequence in a fully automated process. After the chain assembly is completed, the product is released from the solid phase to the solution, deprotected and collected. The presence of side reactions imposes a practical limit on the length of the synthesized 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 with high purity. (Fotin-Mleczek Mariola et al., CN108064176A)
RNA in vitro transcription: the term “RNA in vitro transcription” or “in vitro transcription” relates to the process in which RNA is synthesized (in vitro) in a cell-free system. DNA, in particular plasmid DNA, is used as a template for producing RNA transcripts. RNA can be obtained by DNA-dependent in vitro transcription of a suitable DNA template, which is preferably a linearized plasmid DNA template according to the present invention. The promoter used to control transcription in vivo may be any promoter used for any DNA-dependent RNA polymerase. Specific examples of DNA-dependent RNA polymerases are T7, T3, and SP6RNA polymerases. DNA templates for in vitro RNA transcription can be obtained by cloning a nucleic acid (particularly a cDNA) corresponding to a corresponding RNA to be transcribed in vitro and introducing it into a suitable vector for in vitro transcription (e.g., introducing into the plasmid DNA) . In a preferred specific embodiment of the present invention, the DNA template is linearized with a suitable restriction enzyme and subsequently transcribed in vitro. cDNA can be obtained by reverse transcription or chemical synthesis of mRNA. In addition, DNA templates for RNA synthesis in vitro can also be obtained by gene synthesis. (Fotin-Mleczek Mariola et al., CN108064176A)
Methods for in vitro transcription are known in the art (refer to, for example,  Geall et al. (2013) Semin. Immunol. 25 (2) : 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101-14) .
RNA, mRNA: RNA is a common abbreviation for ribonucleic acid. It is a nucleic acid molecule, that is, a polymer composed of nucleotide monomers. These nucleotides are typically monomers of adenosine monophosphate (AMP) , uridine monophosphate (UMP) , guanosine monophosphate (GMP) and cytidine monophosphate (CMP) , or analogues thereof, which are linked to each other along a so-called skeleton. The skeleton is formed by a phosphodiester bond between the sugar (i.e., ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. The specific order of monomers, i.e., the order of bases attached to the carbohydrate/phosphate skeleton, is called an RNA sequence. Generally, RNA can be obtained by transcription of DNA sequence (for example, in cells) . In eukaryotic cells, transcription typically takes place in the nucleus or mitochondria. In vivo, DNA transcription usually produces so-called premature RNA (also known as pre-mRNA, precursor mRNA or heterologous nuclear RNA) , which must be processed into so-called messenger RNA (usually abbreviated as mRNA) . For example, the processing of premature RNA in eukaryotic organisms includes a variety of different post-transcriptional modifications, such as splicing, 5′-capping, polyadenylation, derivation from nucleus or mitochondria, and similar modifications. The sum of these processes is also called RNA maturation. Mature messenger RNA usually provides nucleotide sequences that can be translated into amino acid sequences of specific peptides or proteins. Typically, the mature  mRNA includes a 5′-cap, optional 5′UTR, an open reading frame, optional 3′UTR, and a poly (A) tail (Fotin-Mleczek Mariola et al., CN108064176A) .
In addition to messenger RNA, there are several non-coding types of RNA, which may be involved in the regulation of transcription and/or translation and immune stimulation. Within the present invention, the term “RNA” further includes any type of single-stranded (ssRNA) or double-stranded RNA (dsRNA) molecule known in the art, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA) , antisense RNA (asRNA) , circular RNA (circRNA) , ribozyme, aptamer, riboswitch, immunostimulating/immunostimulatory RNA, transfer RNA (tRNA) , ribosomal RNA (rRNA) , small nucleolar RNA (snRNA) , small nucleolar RNA (snoRNA) , micro RNA (miRNA) , and Piwi interacting RNA (piRNA) (Fotin-Mleczek Mariola et al., CN108064176A) .
The term “chemically modified” refers to the modification of A, G, U or C ribonucleotides. In general, these terms are not intended to refer to the modifications of ribonucleotide at the naturally occurring 5′end mRNA cap portion. Modifications may be various modifications. In some embodiments, wherein the nucleic acid is an mRNA, the coding region, flanking region and/or terminal region may include one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide introduced into the cell may exhibit reduced degradation in the cell as compared to the unmodified polynucleotide.
As used herein, the term “amino acid” refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxyl group of -CO 2H or a sulfo group of -SO 3H) , wherein an amino acid is linked to a parent molecular group by the side chain, the amino group, or the acid group (e.g., a side chain) . In some embodiments, amino acids are linked to parent molecular groups through carbonyl groups, wherein side chains or amino groups are linked to the carbonyl groups. Exemplary side chains include optionally substituted alkyl, aryl, heterocyclic, alkaryl, alkylheterocyclic, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolidine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine and valine. The amino acid group may optionally be 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 groups, as defined herein (e.g., unsubstituted amino groups) .
Delivery: as used herein, “delivery” refers to the act or manner of delivering a compound, substance, entity, part, cargo or payload.
Delivery Agent: as used herein, “delivery agent” refers to any substance that at least partially promotes in vivo delivery of polynucleotides to target cells.
Expression: as used herein, “expression” of a nucleic acid sequence refers to  one or more of the following events: (1) generation of an RNA template from a DNA sequence (e.g., by transcription) ; (2) processing of RNA transcripts (e.g., by cutting, editing, 5′ cap formation and/or 3′ terminal processing) ; (3) translation of RNA into peptides or proteins; and (4) post-translational modification ofpeptides or proteins.
Formulation: as used herein, “formulation” includes at least one polynucleotide and a delivery agent.
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 in an organism (e.g., an animal, plant, or microorganism) .
In vivo: as used herein, the term “in vivo” refers to events occurring within an organism (e.g., an animal, plant, or microorganism, or its cells or tissues) . Modified: as used herein, “modified” refers to a changed state or structure of a molecule according to the present invention. Molecules can be modified by many methods, including chemical, structural and functional modifications. In one embodiment, the mRNA molecules of the present invention are modified by the introduction of non-natural nucleosides and/or nucleotides, for example, when they relate to natural ribonucleotides A, U, G and C. Atypical nucleotides such as cap structures are not considered “modified” , although they differ from the chemical structures of A, C, G, and U ribonucleotides.
Naturally occurring: “naturally occurring” as used herein means existing in  nature without artificial assistance.
Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” is used herein to refer to compounds, materials, compositions and/or dosage forms that, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
Preventing: as used herein, the term “preventing” refers to partial or complete delay in the onset of an infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, features or clinical manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, features or manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the progression of an infection, particular disease, disorder and/or condition; and/or a reduction of the risk of pathology associated with an infection, disease, disorder and/or condition.
Treating: as used herein, the term “treating” refers to partially or completely alleviating, improving, ameliorating and lightening a particular infection, disease, disorder and/or condition; partially or completely delaying the onset of a particular infection, disease, disorder and/or condition; partially or completely inhibiting the progression of a particular infection, diseases, condition and/or  condition; partially or completely reducing the severity of a particular infection, disease, disorder and/or condition; and/or reducing the incidence of one or more symptoms or features of a particular infection, disease, disorder and/or condition. For example, “treating” cancer may refer to inhibiting the survival, growth and/or spread of tumors. For the purpose of reducing the risk of developing pathology associated with a disease, disorder and/or condition, the treating may be administered to subjects who show no signs of the disease, disorder and/or condition and/or subjects who show only early signs of the disease, disorder and/or condition.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
In order to enable those skilled in the art to better understand the technical aspects of the present invention, some non-limiting embodiments are further disclosed below to provide a further detailed description of the present invention.
The reagents used in the present invention are commercially available or can be prepared by the methods described in the present invention.
The amino acid sequence of the peptide synthesized by the embodiments of the present invention:
TABLE 1 The peptide of the general formula of the present invention
Figure PCTCN2021116359-appb-000010
Figure PCTCN2021116359-appb-000011
Figure PCTCN2021116359-appb-000012
Figure PCTCN2021116359-appb-000013
Table 1 illustrates various peptide sequences that may be used to practice the present invention, but it will 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 explicitly written sequences. On the contrary, it will be readily apparent to such persons that based on the teachings set forth above with respect to the peptides described in the general formula, there may be a large number of peptides potentially suitable for practicing the present invention as set forth herein. Furthermore, it is well within the knowledge of those skilled in the art to determine whether a given peptide sequence falls within the scope of the present invention without the need for improper experimentations using standard techniques in the art. Furthermore, it should be understood that various variants of the peptide sequences appearing in Table 1 are also within the scope of the present invention, as long as such variants satisfy the structural and functional characteristics as set forth above. Variants of the peptide sequence appearing in Table 1 or of any other candidate peptide not explicitly described in Table 1 but satisfying the structural and functional specific requirements as set forth above may include deletion, insertion, substitution using naturally occurring or non-protein amino acids.
Example I: preparation method of the peptide of the present invention
The non-naturally occurring peptides of the present invention are produced by any previously known peptide synthesis methods 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. The solid phase synthesis method (Marrifield, Journal of the American Chemical Society (J. Am. Chem. Soc. ) , 85, 2149-2154, 1963) may be labeled as only one example of such a peptide synthesis method. Currently, peptides can be produced simply and in a relatively short period of time using automated universal peptide synthesizers based on those principles. Additionally, peptides may be produced using well-known recombinant protein production techniques which are widely known to those skilled in the art.
The simple synthesis method and specific process of the peptide of the present invention are described as follows (taking sequence Seq. 05 as an example) :
(1) Resin treatment
① Resin swelling: selecting Fmoc-Arg (pbf) -2-Chlorotrityl Resin (molar substitution coefficient 0.317 mmol/g) as a starting resin and adding into a 50ml reaction column, adding DCM (dichloromethane) for soaking, and draining to complete the swelling of the resin.
② Deprotecting: adding DMF (N, N-dimethylformamide) solution  containing 20% (g/100ml) piperidine, introducing N2 (nitrogen) and stirring for 30 minutes, and filtering out the solvent; and then washing the resin with DMF (N, N-dimethylformamide) for 6 times and draining to complete the deprotecting of the resin.
(2) Amino acid coupling reaction
Reaction monitoring and detection method: Ninhydrin method is used to monitor the reaction process.
The raw materials and reagents used are as follows:
Figure PCTCN2021116359-appb-000014
The specific operation process is as follows:
weighing the corresponding amount of TBTU and protective amino acids in a beaker, adding DMF to dissolve; then adding the reaction solution into the resin deprotected in step (1) , then adding DIEA, introducing N 2 and bubbling for about 90 minutes, and performing the detection with the ninhydrin reaction. After finishing the reaction, the solvent is removed and the resin is washed with DMF  for 3 times. Then, DMF solution containing 20% (g/100ml) piperidine is added into the resin, N 2 is introduced to continue bubbling for 30 minutes, then the solvent is removed, and the resin is washed with DMF for 6 times, thus finishing the coupling of amino acids.
Repeating the above reaction procedure until the condensation reactions of all the protected amino acids are finished. After coupling to the last protected amino acid, the peptide is contracted, washed with DMF, DCM and methanol for three times in turn, drained and weighed.
Condensation method: TBTU + DIEA, condensation agent: TBTU: 0.64 g, DIEA: 0.7 ml.
Table 2: Abbreviations of amino acids and corresponding protected amino acidspeptide
Figure PCTCN2021116359-appb-000015
Figure PCTCN2021116359-appb-000016
(3) TFA cleavage (dissociation of peptide from resin and removal of amino acid side chain protecting group)
adding resin finally obtained from " (2) Amino acid coupling reaction" into the prepared pyrolysis solution (containing 86%TFA, 5%EDT (Ethanedithiol) , 5%benzene methyl sulfide, 3%phenol and 2%pure water) and stirring for 150 minutes; then separating the resin from the lysate, adding ether into the separated lysate to fully precipitate the peptide; and filtering, and washing with ether for 6 times to obtain the crude peptide, purifying to obtain the peptide shown in sequence of Seq. 05. By analogy, other peptides provided by the present invention  are synthesized.
(4) Simple steps of purification
Weighing the crude product obtained in step (3) and dissolving with 3ml of a mixture of acetonitrile and water with a volume ratio of 1∶1, clarifying and filtering, and detecting the purity and molecular weight of peptide by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) . The results are shown in Table 1, and the purity ofpeptides is over 98.00%.
Example II: preparation of peptide complex nanoparticles
The peptide complex nanoparticle of the present invention can be prepared by referring to the nanoparticle preparation method of patents CN111249476A, CN111281981B, CN111281982A, CN111285845B, CN111588637A or an application numbered 202110713076.9.
Preparation method I: dissolving the peptide in the nuclease-free ultrapure water to obtain a solution of 1 mg/ml, then mixing the peptide and mRNA according to the mass ratio in Table 3 and stirring for 15 minutes, and then standing to obtain the peptide complex nanoparticles. The specific prescriptions are shown in Table 3:
Table 3. Prescription, particle size, potential and encapsulation rate of peptide nanoparticles
Figure PCTCN2021116359-appb-000017
Preparation method II: according to the prescription design in Table 4, taking PEG derivative auxiliary materials 304, and/or 90R4, and/or 17R4, and/or T90R4R, and/or 704, and/or L64 from a refrigerator at -20℃, balancing to 25 ℃, weighing at 25 ℃, dissolving in the nuclease-free ultrapure water in a nuclease-free 15 ml centrifuge tube to make its concentration of 100 mg/ml, and fully vortexing with a vortexer for 5 minutes to obtain a stock solution A. At 25 ℃, taking the lipid auxiliary material DSPC or PC from the refrigerator at -20℃ and balancing to 25 ℃, weighing at 25 ℃, dissolving in ethanol (chromatographic grade) in a nuclease-free 1.5 ml centrifuge tube at 25 ℃ to make its concentration of 10 mg/ml, and fully vortexing with a vortexer for 5 minutes to obtain a stock solution B.
Dissolving the corresponding peptides in the table into a solution of 5 mg/ml  by adding nuclease-free ultra-pure water, and then mixing with mRNA at the mass ratio shown in Table 4 for 10 minutes successively to obtain a stock solution C, mixing the stock solution A and the stock solution B for 1 minute according to the mass ratio shown in Table 4, then adding the stock solution C, and after fully vortexing with a vortexer for 1 minutes to obtain the peptide complex nanoparticles. The specific prescriptions are shown in Table 4:
Table 4. Prescription, particle size, potential and encapsulation rate of peptide nanoparticles.
Figure PCTCN2021116359-appb-000018
Preparation method III: weighing 5.0 mg of DMG-PEG and 1 ml of chromatographic pure ethanol for dissolution, weighing 10.3 mg of DSPC (distearoyl phosphatidylcholine) and adding 1 ml of chromatographic pure ethanol for dissolution; and weighing 19.4 mg of cholesterol and adding 2ml of chromatographic pure ethanol for dissolution.
Dissolving the peptide in the nuclease-free ultrapure water to obtain a solution of 1 mg/ml, then mixing the peptide with mRNA uniformly in turn according to the corresponding mass ratio in Table 5, after 5 minutes, adding 20 ml of citrate buffer with a pH value of 5.4, stirring continuously at 1500 rpm, adding ethanol solution of DMG-PEG and DSPC dropwise into the citrate buffer, then adding some volume of ethanol solution of cholesterol with concentration of  19.4 mg/ml dropwise, such as 100uL, 200uL, 250uL or 500 uL, stirring continuously for 30 minutes, and removing ethanol under reduced pressure at 40 ℃ to obtain the peptide complex nanoparticles. The specific prescriptions are shown in Table 5:
Table 5. Prescription, particle size, potential and encapsulation rate of peptide nanoparticles
Figure PCTCN2021116359-appb-000019
Preparation method IV: weighing 10 mg of a corresponding auxiliary material, such as DMG-PEG, PEG-CLS or PEG-DSPE in Table 6, dissolving the material by adding the nuclease-free ultrapure water to make its concentration of 1 mg/ml, so as to obtain the stock solution A. Dissolving the peptide in the nuclease-free ultrapure water to obtain a solution of 1 mg/ml, so as to obtain a stock solution B. After the stock solution A and the stock solution B are mixed for 1 minute according to the corresponding mass ratio in Table 6, the mixed solution and mRNA are respectively mixed for 10 minutes, 15min, 30min or 60min, and the peptide complex nanoparticles are obtained by fully vortexing with a vortexer for 20 minutes. The specific prescriptions are shown in Table 6:
Table 6. Prescription, particle size, potential and encapsulation rate of peptide nanoparticles.
Figure PCTCN2021116359-appb-000020
The prepared aqueous solution of the peptide complex nanoparticles is mixed with a lyophilized agent, and then lyophilized by a lyophilizer (Christ Alpha LD plus, Germany) to prepare a lyophilized agent, which could be trehalose or sucrose, and stored in a refrigerator at 4℃ for later use.
Example III: characterization of the peptide complex nanoparticles of the present invention
Nanoparticle morphology: EGFP-mRNA is used as model mRNA, the peptide complex nanoparticle is prepared according to the preparation method I, or the preparation method II, or the preparation method III, or the preparation method IV and the corresponding prescriptions in Example II, and the nanoparticle morphology of the representative peptide complex nanoparticle aqueous solutions of the present invention is tested by using a transmission electron microscope (model FEI Talos F200X) . The copper grid without any dyeing is immersed in the freshly prepared aqueous solution of peptide complex nanoparticles, and naturally dried at 25 ℃ to prepare samples, which are obtained by testing. As shown in FIG. 1, the results show that the peptide complex nanoparticles of the present invention have good dispersibility, present regular or  irregular spherical structure, and the particle size ranges from 60 nm to 120 nm. The results are shown in FIG. 1.
Particle size and potential: EGFP-mRNA is used as model mRNA, the peptide complex nanoparticles are prepared according to the preparation methods described in Example II, and the dynamic light scattering particle size, Zeta potential and polydispersity (PDI) of the peptide complex nanoparticles are measured by Malvern Zetasizer Nano ZSE at 25 ℃. The results are shown in Tables 3 to 6. The results show that the peptide complex nanoparticles have a size ranges from 56 nm to 273 nm, with good dispersibility, and the surface charge of the nanoparticles is between -15 mV and 5 mV.
Encapsulation rate: FLuc-mRNA is used as model mRNA, the peptide complex nanoparticles are prepared according to the preparation methods described in Example II, Quant-iT RiboGreen RNA detection kit (available from ThermoFische Company) is used to determine the mRNA encapsulation rate of each prescription. For the specific method, referring to the instruction of the kit. The brief treatment method of the present invention is as follows: centrifuging each prescription at 4℃ and 20000 rpm for 2 hours by using a low-temperature high-speed centrifuge, collecting the supernatant and quantify its volume with a pipette, and recording the volume as V1; using Quant-iT RiboGreen RNA detection kit to measure the concentration of mRNA in the supernatant, recoding the concentration as C1; dissolving the centrifuged precipitate by adding 25 ul of chromatographic pure DMSO, continuing to add 0.9%normal saline injection to  mix evenly, after standing at 25 ℃ for 2 hours, recording the total volume as V2, and using Quant-iT RiboGreen RNA detection kit to measure the concentration of mRNA, recording the concentration as C2; the formula for calculating the entrapment rate of each prescription is as follows: entrapment rate = 100%-(V1C1) / (V1C1+V2C2) × 100%. The results are shown in Table 3 to Table 6. All prescriptions have good encapsulation effect on mRNA, and the entrapment rate is above 98.0%.
Example IV: agarose gel electrophoresis for detection of ability of peptides to compress mRNA
Preparing the agarose gel with a mass to volume ratio of 1% (agarose 0.4 g: 1×TAE buffer 40 ml) , microwaving twice to melt it fully, adding 4 μl SyBR Safe DNA Gel Stain dye (Lot No. 1771519, Invitrogen, USA) to the agarose at a ratio of 1∶10000, mixing well and pouring into the corresponding gel tank (15-well slot) and cooling for 20 minutes before use.
Configuration method of mRNA positive control group: EGFP-mRNA is used as model mRNA, adding 1 μl mRNA solution with a concentration of 100 ng/μl (i.e., 100 ng) , then adding 9 μl nuclease-free ultrapure water to make up the volume of the system to 10 μl, and finally adding 2 μl of 6*loading buffer to mix evenly.
Sample set configuration method: according to different mass ratios of peptide to mRNA, 1 μl of mRNA solution with a concentration of 100 ng/ul (i.e., 100ng) is added into peptide solution (1 μg/μl) and mixed evenly, then nuclease- free ultrapure water is added to make up the volume of the system to 10 μl, after mixing for 10 minutes, 2 μl of 6*loading buffer is added to each sample for uniform mixing. After the samples are mixed, 12 μl system is added to each well, and the gel is run for 25 minutes by 80V voltage electrophoresis instrument, and observed by gel imager. The experimental results are shown in FIG. 2.
Conclusion: the minimum mass ratio (peptide: mRNA) of each peptide to ensure that mRNA is fully compressed is: 4 for Seq. 05, 4 for Seq. 12, 2 for Seq. 46, 2 for Seq. 47, 16 for Seq. 49 and 4 for Seq. 53.
Example V: in vitro cell transfecfion experiment and cytotoxicity investigation of peptide complex nanoparticles
Cell transfection mRNA: FLuc-mRNA is used as model mRNA, DC2.4 cell suspension in logarithmic growth phase is aliquoted into 96-well plate at the density of 4 × 10 4 cells per well, and then put into 37℃, 5%CO2 incubator for static culture. After 24 hours, FLuc-mRNA with a concentration of 1 μg/μl is diluted to 0.1 μg/μl in nuclease-free ultrapure water, and take FLuc-mRNA to prepare peptide complex nanoparticles according to the preparation method of different prescriptions described in Example 2, and then respectively dilute them with nuclease-free ultrapure water to 88μl of peptide nanoparticle composition containing 10ng/μl FLuc-mRNA liquidpeptide, after standing for 10 minutes, the mixture is added to 96-well plate containing 180 μl opti-MEM culture medium per well at a volume of 20 μl per well, and each sample is repeated for 4 wells.  After 4 hours of administration, the culture solution in 96-well plate is replaced with complete culture medium. Continuing to culture for 24 hours, the complete culture medium is aspirated and washed with PBS, 100 μl D-Luciferin working solution (working concentration: 250 μg/ml) is added to each 96-well plate, and then cultured in 37 ℃ incubator for 5 minutes, the fluorescence expression intensity of FLuc-mRNA is measured by Omega-Fluostar microplate reader. The results are shown in FIG. 3.
Cytotoxicity experiment: DC2.4 cell suspension in logarithmic growth phase is aliquoted into 96-well plate at the density of 4 × 10 4 cells per well, and then put into 37 ℃, 5%CO 2 incubator for static culture. After 24 hours, FLuc-mRNA with a concentration of 1 μg/μl is diluted to 0.1 μg/μl in nuclease-free ultrapure water, and take FLuc-mRNA to prepare peptide complex nanoparticles according to the preparation method of different prescriptions described in Example 2, and then respectively dilute them with nuclease-free ultrapure water to 88μl of peptide nanoparticle composition containing 10ng/μl FLue-mRNA liquidpeptide, after standing for l0 minutes, it is added to 96-well plate containing 180 μl opti-MEM per well at a volume of 20 μl per well, and each sample is repeated for 4 wells. After 4 hours of administration, the culture solution in 96-well plate is replaced with complete culture medium. Continuing to culture for 48 hours, the complete culture medium is aspirated and washed with PBS for three times, the cell pores without prescription are used as negative control, and the cell-free CCK-8 medium pores are used as blank control, 90 μl serum-free culture medium and l0  μl CCK-8 solution are added to each well, and the incubation is continued for 2 hours in the incubator. The absorbance at 450 nm is measured by Omega-FLuostar microplate reader. Calculation formula of cell viability:
Cell viability *%= [A (adding medicine) -A (blank) ] / [A (without adding medicine) -A (blank) ] × 100%
A (adding medicine) : the absorbance of each well added with DC2.4 cells, prescription solution and CCK-8 solution
A (blank) : the absorbance of each well only added with CCK-8 solution
A (without adding medicine) : the absorbance of each well added with DC2.4 cells and CCK-8 solution
*Cell viability: cell proliferative activity or cytotoxic activity.
The results are shown in FIG. 4.
Conclusion: The results show that the survival rate of cells is above 90%, which indicates that the prescription of peptide complex nanoparticles has no obvious cytotoxicity, has good biocompatibility, and can be used for subsequent experiments in animals.
Cell transfection of DNA: Luc-pDNA is used as model mRNA, DC2.4 cell suspension in logarithmic growth phase is aliquoted into 96-well plate at the density of 4 × 10 4 cells per well, and then put into 37℃, 5%CO 2 incubator for static culture. After 24 hours, Luc-pDNA with a concentration of 1 μg/μl is diluted to 0.1 μg/μl with the nuclease-free ultrapure water. take Luc-pDNA to prepare peptide complex nanoparticles according to the preparation method of different  prescriptions described in Example 2, and then respectively dilute them with nuclease-free ultrapure water to 88μl of peptide nanoparticle composition containing 15ng/μl Luc-pDNA liquid, after standing for 30 minutes, the mixture is added to 96-well plate containing 180 μl opti-MEM culture medium per well at a volume of 20 μl per well, and each sample is repeated for 4 wells. After 4 hours of administration, the culture solution in 96-well plate is replaced with complete culture medium. Continuing to culture for 24 hours, the complete culture medium is aspirated, and 100 μl D-Luciferin solution with a working concentration of 250 μg/ml is added into each 96-well plate, and then cultured in incubator at 37 ℃ for 5 minutes, finally, the fluorescence expression intensity of Luc-pDNA is tested by imaging with an Omega-FLuostar microplate reader, the test is repeated every 24 hours, the medium containing D-Luciferin is aspirated after each test, fresh complete culture medium is added to continue culturing for 24 hours, and then D-Luciferin is added for testing, repeated for 3 days. Results are shown in FIG. 5, the abscissa represents different prescriptions, and the ordinate shows a relative fluorescence intensity expressed by Luc-pDNA with the same dose after transfection for 24h, 48h and 72h. The results are shown in FIG. 5.
Conclusion: as shown in FIG. 5, the peptide complex nanoparticles encapsulated with Luc-pDNA shows good expression at cellular level, with the highest expression on the second day and decreasing from the third day, wherein Rp.01, Rp. 27 and Rp. 34 are superior to other prescriptions.
Example VI: detection of in vivo transfection of peptide complex  nanoparticles in mice by small animal fluorescence imaging
Three female BALB/c mice in each group, FLuc-mRNA is used as model mRNA, and preparing a peptide complex nanoparticles prescription containing FLuc-mRNA through the method of the present invention. In the experimental group, each mouse is injected with 50 μl peptide complex nanoparticles prescription containing 5 μg FLuc-mRNA by insulin needle. The administration mode of recipe groups Rp. 01, Rp. 07, Rp. 11, Rp. 12, Rp. 13, Rp. 17, Rp. 20, Rp. 22 and Rp. 24 is subcutaneous injection, and the injection site is subcutaneous in the back of mice; the administration mode of recipe groups Rp. 33, Rp. 36, Rp. 37 and Rp. 42 are intraperitoneal injection; and the administration mode of other recipe groups is intramuscular injection, and the injection site is the thigh muscle of mice. The blank control group is represented by NC, and 50 μl PBS buffer is injected intramuscularly with the insulin needle. After 6 hours of administration, a proper amount of D-Luciferin is diluted with PBS to prepare a solution with a concentration of 25 mg/ml, keeping away light and spare, each mouse is intraperitoneally injected with 125 μl substrate, and the mice are placed in a small animal anesthesia box, and a vent valve is opened to release isoflurane to anesthetize the mice. Five minutes after substrate injection, whole body in-vivo imaging bioluminescence images of mice are detected by small animal in-vivo imaging system (PerkinElmer, IVIS Lumina Series III) . Recipe groups Rp. 33, Rp. 36, Rp. 37, Rp. 42 are the captured abdominal bioluminescence images of the mice, and other recipe groups are the captured back bioluminescence images.  Results are shown in FIG. 6, a representative mouse is selected for each group, in the experimental group, the prescription ofpeptide complex nanoparticles showed luciferase expression in whole body in-vivo imaging, the greater the fluorescence intensity, the more luciferase expression.
Conclusion: as shown in FIG. 6, the peptide complex nanoparticles encapsulated with FLuc-mRNA in each experimental group have good luciferase expression in mice. Also, intraperitoneal, subcutaneous and intramuscular injections are effectively expressed. Luciferase in Rp. 27, Rp. 33 and Rp. 41 are superior to other recipe groups in the experimental group.
Example VII: evaluation of humoral immune effect of peptide complex nanoparticles in mice
Novel coronavirus S-mRNA is used as model mRNA, which is provided by Shanghai Hongene Biotech Corporation. The nucleotide sequence of the novel coronavirus S-mRNA (cap1 structure, N1-me-pseudo U Modified) is shown in the sequence table for S-mRNA.
The specific information of S-mRNA stock solution is as follows:
Product name: COVID-19 Spike Protein, Full Length-mRNA;
Product description: 4088 nucleotide in length;
Modifications: Fully substituted with N1-Me-pseudo UTP; (All replaced with N1-Me-pseudo UTP)
Concentration: 1.0 mg/ml;
Storage environment: 1 mM sodium citrate at pH 6.4;
Storage requirements: -40℃ or below.
Experimental process:
Step 1: first immunization in mice: on day 0, female BALB/c mice at 5-6 weeks are divided into 9 groups (5 mice per group) and injected intramuscularly with 75 μl PBS (blank control) , 5 μg combination of naked S-mRNA and 5 μg S protein (positive control) and 75 μl peptide complex nanoparticle recipe groups Rp. 21, Rp. 25, Rp. 27, Rp. 41, Rp. 01, RP. 08 encapsulated with 5 μg S-mRNA.
Step 2: first serum collection: on the 28th day, blood is collected from the outer canthus of mice. The serum is solidified at 4 ℃ for 1 hour, then centrifuged at 5000× g at 4℃ for 5 minutes, removing the supernatant, and then centrifuged at 10000× g at 4℃ for 5 minutes, removing the supernatant, aliquoted by adding into eight consecutive PCR tubes, and frozen at -20℃ for later use.
Step 3: second immunization in mice: on the 28th day, after blood collection from the outer canthus, the mice is injected intramuscularly with 75 μl PBS (blank control) , 5 μg combination of naked S-mRNA and 5 μg S protein (positive control) and 75 μl peptide complex nanoparticle recipe groups Rp. 21, Rp. 25, Rp. 27, Rp. 41, Rp. 01, RP. 08 encapsulated with 5 μg S-mRNA. Repeating the first immunization process.
Step 4: second serum collection: 21 days after the second immunization,  blood is collected from the outer canthus of mice. The serum is solidified at 4 ℃ for 1 hour, then centrifuged at 5000× g (5000 times gravity acceleration) at 4 ℃ for 5 minutes, removing the supernatant, and then centrifuged at 10000 × g at 4℃ for 5 minutes, removing the supernatant, aliquoted by adding into eight consecutive PCR tubes, and frozen at -20℃ for later use.
Step 5: detecting serum IgG content by ELISA: the S protein is diluted in PBS and the ELISAplate is coated with 100 μl dilution (containing 1 μg S protein) per well for 6 hours at 4℃. The liquid in the plate is discarded, 200 μl PBST is added to each well to wash the plate for 3 times, and then 200 μl PBS blocking solution containing 5%BSA is added into each well to seal with shaking table at 25℃ for 2 hours. The blocking solution is discarded, the plate is washed once with 200 μl PBST per well, then 100 μl serum diluted 200 times with PBS is added, and incubated in the shaking table at 25 ℃ for 2 hours. The serum is discarded, the plate is washed 3 times with 200 μl PBST per well, and then 100 μl antibody (diluted with PBS at a ratio of 1∶1000) is added to each well, and incubated in the shaking table at 25 ℃ for 1 hour. After the antibody is discarded, the plate is washed with 200 μl PBST for three times, and then 50 μl TMB substrate is added to avoid light. After the positive control well turned dark blue or the reaction lasted for 10 minutes, 5 μl 2M sulfuric acid is added to stop the reaction. The optical density at the wavelengths of 450 nm and 630 nm wavelengths is detected by ELIASA, and the difference of OD values is calculated to reflect the level of anti-S protein IgG in the serum. The results are shown in FIG. 7.
Summary: the results showed that the OD value corresponding to the recipe group Rpl4 is higher than that of the control group after the two immunizations, suggesting that the prescription nanoparticles had strong serum conversion efficiency and humoral immune activation function.
Step 6: serum IgG titer detected by ELISA: the S protein is diluted in PBS and the ELISA plate is coated with 100 μl dilution (containing 1 μg S protein) per well for 6 hours at 4℃. The liquid in the plate is discarded, 200 μl PBST is added to each well to wash the plate for 1 times, and then 200 μl PBS blocking solution containing 5%BSA is added into each well to seal with the shaking table at 25 ℃ for 2 hours. The blocking solution is discarded, the plate is washed three times with 200 μl PBST per well, then the serum diluted 50, 250, 1250, 6250, 31250, 156250, 781250 and 3906250 times with PBS at a ratio of 1∶3 are added, and incubated in the shaking table at 25 ℃ for 2 hours. The serum is discarded, the plate is washed 3 times with 200 μl PBST per well, and then 100 μl antibody (diluted with PBS at a ratio of 1∶1000) is added to each well, and incubated in the shaking table at 25 ℃ for 1 hour. After the antibody is discarded, the plate is washed with 200 μl PBST for three times, and then 50 μl TMB substrate is added to avoid light. After the positive control well turned dark blue or the reaction lasted for 10 minutes, 5 μl 2M sulfuric acid is added to stop the reaction. The optical density at 450 nm and 630 nm is detected by ELIASA. The results are shown in FIG. 8.
Summary: the present invention takes 2 times of the average OD value of  PBS group as the baseline, and the OD value of Rp. 08 group is still 2 times higher than the baseline when diluted to 3906250 times, suggesting that the recipe group Rp.08 has strong serum transformation efficiency and humoral immune activation function.
Example VIII: gene transfection kit
The gene transfection kit is a versatile transfection reagent that can be composed of any of the prescriptions of the present invention and provides efficient transfection in a variety of adherent and suspension cell lines. It is suitable for all common cell lines and many cell lines that are difficult to transfect, and can be used in medium containing or not containing serum. The kit of the present invention is used to transfect mammalian cells with a 96-well cell culture plate. The specific steps are as follows:
1. On the day before transfection, Inoculate 1 × 10 4 to 10 × 10 4 cells on a 96-well cell culture plate with 200μl medium per well, so that the growth density of cells reached more than 80%during transfection.
2. For each transfection sample, the following complexes are prepared:
a. diluting 200 ng DNA or RNA to 15 μl with sterile nuclease-free water and mixing gently;
b. gently mixing the transfection reagent according to the prescription ratio before use, and then diluting the appropriate amount to 15 μl with sterile nuclease-free water; and
c. gently mixing the diluted DNA or RNA with diluted transfection reagent according to prescription ratio (total volume = 30 μl) , and incubating at 25 ℃ for 10 minutes to 30 minutes, get nucleic acid-peptide complex nanoparticles.
3. After the 96-well plate is rinsed with PBS, 170 μl opti-MEM medium is added to each, then 30 μl nucleic acid-peptide complex nanoparticles are added, and the final volume of the medium is 200μl.
4. The cells are incubated in a CO 2 incubator and the original medium is replaced with complete medium after 4 hours, the incubation is continued in the CO 2 incubator for 12 hours to 72 hours, finally an expression amount of the nucleic acids is detected.
All references disclosed herein, including patent documents, are incorporated by reference. The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the present invention which may be subject to various modifications and variations to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims (14)

  1. A peptide compound having a following structure of general formula I:
    (Xaa)  x-Arg-Val-Gln-Pro-Thr-Glu-Ser-Ile-Val-Arg- (Yaa)  y  (General Formula I)
    wherein: x is an integer from 1 to 25 and y is an integer from 0 to 10; and
    (Yaa) y is a peptide segment consisting of any amino acid, (Yaa) y is a peptide segment consisting of any amino acid.
  2. The peptide compound according to claim 1, wherein the Xaa is selected from at least one of Arg, Trp, Cys, Lys, Leu, Phe, Pro or His; and/or the Yaa is selected from at least one of Arg, Trp, Phe and Cys.
  3. The peptide compound according to any one of claims 1 to 2, wherein the (Xaa) x consists of Arg; or
    the (Xaa)  x is (Xa′a′)  n (Arg)  1-10 (Xa′a′)  n, where Xa′a′ is selected from Arg, Trp, Cys, Lys, Leu, Phe, Pro, or His, and n is an integer from 0 to 10.
  4. The peptide compound according to any one of claims 1 to 3, wherein an amino acid sequence of the peptide 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.
  5. The peptide compound according to claim 1 or 4, wherein the general  formula (I) is at least 50%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 20%; or
    the general formula (I) is at least 75%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 50%; or
    the general formula (I) is at least 90%similar to any one of Seq. 01 to Seq. 53 and improves delivery of nucleic acid molecules into cells by at least 100%; or
    the general formula (I) is at least 90% similar to any one of RRRRRWCRVQPTESIVR, RRRRRWFCRVQPTESIVR, FCRWCRRVQPTESI VRRCWRCF, FCRWCRRVQPTESIVCWRRRCF, HKRWCRRWCRVQPTESIV RC or WCRRRVQPTESIVRRRWC.
  6. A peptide complex nanoparticle composition, which comprises:
    a) at least one of the peptide compound of any one of claims 1 to 5; and
    b) a nucleic acid.
  7. The peptide complex nanoparticle of claim 6, wherein the nucleic acid is chemically modified or unmodified DNA, single-stranded or double-stranded DNA, coding or non-coding DNA, or the nucleic acid may be chemically modified or unmodified RNA, single-stranded or double-stranded RNA, coding or non-coding RNA.
  8. The peptide complex nanoparticle of claim 7, wherein the nucleic acid is mRNA.
  9. The peptide complex nanoparticle of claim 6, which is characterized in  that the peptide complex nanoparticle further comprises a composition of at least one auxiliary material.
  10. The peptide complex nanoparticle of any one of claim 6-9, the mass ratio of the nucleic acid to the peptide is less than or equal to 1∶1, and/or the mass ratio of the nucleic acid to the auxiliary materiale is less than or equal to 1∶2.
  11. The peptide complex nanoparticle of claim 9, which is characterized in that the auxiliary material is selected from one or more of lipids and PEG derivatives.
  12. A peptide complex nanoparticle composition, which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 05 and auxiliary materials, the auxiliary materials are
    Figure PCTCN2021116359-appb-100001
    and lecithin, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 05, 
    Figure PCTCN2021116359-appb-100002
    and lecithin is 1∶2∶322∶1 ; or
    which comprises: a nucleic acid, the peptide compound with the amino acid sequence of Seq. 49 and auxiliary materials, the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 49, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol is 10∶ 300∶ 8∶ 16∶ 31; or
    which comprises: a nucleic acid, the peptide compound with the amino acid  sequence of Seq. 53 and auxiliary materials, the auxiliary materials are 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol, the mass ratio of nucleic acid, peptide compound with amino acid sequence of Seq. 53, 1, 2-dimyristoyl-glycerol-3-methoxypolyethylene glycol 2000, 1, 2-distearoyl-sn-glycerol-3-phosphate choline and cholesterol is 10∶ 40∶ 8∶ 16∶ 5.
  13. A nucleic acid vaccine, wherein the nucleic vaccine comprises the peptide complex nanoparticle composition of any one of clams 6 to 12.
  14. A use of the peptide complex nanoparticle of any one of claims 6-12 in preparing medicines or kits.
PCT/CN2021/116359 2020-09-03 2021-09-03 Peptide and peptide complex nanoparticle, nucleic acid vaccine and application thereof WO2022048619A1 (en)

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