Multimeric delivery systems for intracellular delivery of molecules

Abstract

The present invention relates to the field of molecular biology, and more specifically, to a multimeric delivery system that can be used for intracellular delivery of cargo molecules. The multimeric delivery system of the present invention can achieve efficient endocytosis of cargo molecules and efficient release from endocytic vesicles, significantly improving the cytoplasmic delivery efficiency of cargo molecules. Once cargo molecules are available in the cytoplasm, they can play any role associated with them. Therefore, the multimeric delivery system of the present invention provides an effective means for influencing the biological mechanisms and pathways of cells, which can be used in many fields such as research, treatment, and diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and more particularly to a polymerized delivery system for intracellular delivery of cargo molecules. Background Art

[0002] As a selective permeable barrier, the cell membrane is crucial to cell survival and function. Although small molecules can pass through the cell membrane through natural processes of the cell or direct diffusion through the lipid bilayer, in most cases, the effective passage of intracellular cargoes such as exogenous active biomacromolecules through the cytoplasmic membrane is still the main obstacle to the cell transport process. Therefore, a molecular transport tool that can effectively improve the efficiency of transporting intracellular cargoes to living cells is extremely important for its application in biomedicine and other fields.

[0003] At present, the use of cell-penetrating peptides (CPPs) to mediate the entry of biomacromolecules into cells is one of the research hotspots in the field of biomedicine. CPPs are generally polypeptides of 5-30 amino acids, which can mediate biomacromolecules including proteins to pass through the cell membrane and enter the cell through chemical cross-linking, fusion expression or non-covalent binding. Cell-penetrating peptides have been applied to various fields of biomedicine due to their advantages of low dosage, short transport time, controllable dosage, simple operation, low immune response and toxic side effects when mediating the entry of biomacromolecules into cells.

[0004] Although CPPs have many advantages in transducing biomacromolecules, they still have some limitations, mainly including their inherently low delivery efficiency and the further reduced delivery efficiency in the presence of serum.

[0005] Studies have shown that only 1% of the biomacromolecules carried by CPPs can successfully escape from endocytic vesicles, and most of them are eventually directed to lysosomes for degradation. Therefore, the vesicle escape efficiency of intracellular cargo is also a key limiting factor in the intracellular delivery process of CPPs, and its efficiency determines the overall delivery efficiency. A large number of studies have been devoted to solving the problem of low CPPs delivery efficiency. In terms of improving the escape efficiency of endocytic vesicles, the main strategy is to destroy the integrity of the vesicle membrane during the process of maturation and acidification, so that its contents (including the delivered intracellular cargo) are released into the cytoplasm. At present, the most effective way is to use pH sensitive fusogenic peptides derived from viruses, bacteria, animals, plants or humans. pH sensitive peptides contain a certain proportion of hydrophobic amino acids, and their conformation changes dramatically under low pH conditions. After CPPs mediate cellular endocytosis, during the process of maturation and acidification of endocytic vesicles, once the pH value drops to the critical point, the pH-sensitive peptide coupled to CPPs undergoes conformational changes and binds to the lipid bilayer of the vesicle membrane, thereby violently disturbing the integrity of the phospholipid bilayer membrane, forming small holes on it or causing the membrane to lyse, and finally releasing the transported biomacromolecules into the cytoplasm. However, subsequent studies have found that although the endocytic vesicle escape efficiency of the CPPs macromolecular intracellular delivery system after fusion with pH-sensitive peptides is indeed improved, a considerable portion of the transported macromolecules still remain in the endocytic vesicles (with fluorescent protein as the transported macromolecule, obvious dot distribution can be seen).

[0006] Another factor limiting the application of membrane-penetrating peptides is serum intolerance. That is, under high serum concentration conditions, CPPs cannot fully contact the cell membrane due to the electrostatic interaction between their positive charge and the negative charge on hemoglobin, resulting in a sharp drop in their delivery efficiency. This also seriously limits the widespread application of CPPs in vivo.

[0007] In summary, although a lot of work has been done to improve the efficiency of CPPs-mediated macromolecular entry, the results are still unsatisfactory. The efficiency of entry is still the main limiting factor for the widespread application of CPPs in the development of biomacromolecule drugs targeting intracellular targets. At the same time, serum tolerance is also a problem that must be solved for CPPs in drug development, and there are currently no research reports on this issue. Summary of the invention

[0008] After a large number of experiments and repeated explorations, the inventors of this application unexpectedly found that the introduction of a multimerization domain can significantly improve the endocytic efficiency of cargo molecules and significantly improve serum tolerance. In addition, the use of pH-sensitive peptides in conjunction with specific protease recognition sequences can significantly improve the release of cargo molecules from endocytic vesicles, further improving the cytoplasmic delivery efficiency of cargo molecules, allowing cargo molecules to fully exert their corresponding biological functions. Based on these findings, the inventors have developed a multimerization delivery system that can achieve efficient cytoplasmic delivery.

[0009] Fusion peptide

[0010] Therefore, in a first aspect, the present invention provides a fusion polypeptide comprising a multimerization domain sequence, a cell-penetrating peptide, a pH-sensitive fusogenic peptide and a protease recognition sequence.

[0011] In the present invention, the term "multimerization domain" refers to any polypeptide or protein that is capable of multimerizing several copies of the fusion polypeptide described herein (i.e., forming a biomolecular complex). In certain embodiments, the multimerization domain is a homologous multimerization domain that aggregates the same fusion polypeptide and forms a multimer.

[0012] In certain embodiments, the multimerization domain can be a dimerization domain, a trimerization domain, a tetramerization domain, or essentially any higher order multimerization domain so long as the domain is capable of facilitating interactions between at least two domains (and the polypeptides of which they form a part).

[0013] In certain embodiments, the multimerization domain is selected from the group consisting of: leucine zipper, NOE (SEQ ID NO: 3), GCN4-P1 (SEQ ID NO: 4), Delta (SEQ ID NO: 5), and any combination thereof.

[0014] In certain embodiments, the multimerization domain is selected from a leucine zipper.

[0015] In certain embodiments, the multimerization domain sequence comprises the sequence shown in SEQ ID NO:1 or 2.

[0016] In the present invention, the term "pH-sensitive fusogenic peptide" and "pH-sensitive peptide" are used interchangeably, which refers to a class of polypeptides that can undergo conformational changes under acidic conditions (e.g., pH <6.5) to promote fusion with the endocytic vesicle membrane. When the pH-sensitive peptide is endocytosed by the cell, during the maturation and acidification of the endocytic vesicle, once the pH value drops to the critical point, such polypeptides undergo conformational changes and bind to the lipid bilayer of the vesicle membrane, thereby severely disturbing the integrity of the phospholipid bilayer membrane, forming small holes thereon or causing the membrane to lyse, thereby releasing the transported biomacromolecules into the cytoplasm. Such polypeptides are well known in the art and are described in, for example, Varkouhi, Amir K., et al. Journal of Controlled Release 151.3 (2011): 220-228; Erazo-Oliveras A, Muthukrishnan N, Baker R, et al. Pharmaceuticals, 2012, 5(11): 1177-1209, all of which are incorporated herein by reference.

[0017] The pH sensitive peptide that can be used in the fusion protein of the present invention can be selected from the following proteins or polypeptides, or derived from the following proteins or polypeptides:

[0018] Viral protein sources: HA2 (influenza virus) and its mutants KALA, GALA; penton base (adenovirus or rhinovirus), gp41 (HIV), L2 (papillomavirus), envelope protein (West Nile virus);

[0019] Bacterial protein sources: Listeriolysin O (LLO), Pneumococcal pneumolysin (PLO), Streptococcal streptolysin O (SLO), Diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Shiga toxin, Cholera toxin;

[0020] Plant protein sources: Ricin, Saporin, Gelonin toxin;

[0021] Human / animal protein sources: human calcitonin, fibroblast growth factor receptor, melittin;

[0022] Artificially synthesized peptides: (R-Ahx-R)(4)AhxB, Penetratin (pAntp), EB1, Bovine prion protein (bPrPp), Sweet Arrow Peptide (SAP), Poly (L-histidine), Proline-rich peptide (Proline-rich).

[0023] In certain embodiments, the pH sensitive fusion peptide is selected from influenza virus HA2 (SEQ ID NO: 36) or a mutant thereof, melittin (SEQ ID NO: 39), and any combination thereof. In certain embodiments, the mutant of influenza virus HA2 is selected from INF7 (SEQ ID NO: 12), KALA (SEQ ID NO: 37) or GALA (SEQ ID NO: 38).

[0024] In certain embodiments, the pH-sensitive fusion peptide comprises INF7. In certain embodiments, the pH-sensitive fusion peptide comprises or consists of the following sequence: SEQ ID NO:12.

[0025] In the present invention, the term "cell penetrating peptide (CPP)" is also called "cell penetrating peptide", "protein translocation domain (PTD), "Trojan horse peptides" or "transduction peptide", etc., which refers to a polypeptide that can promote the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids). Such polypeptides are well known in the art and are described in, for example, Stewart KM, et al. Org Biomol Chem. 2008 Jul 7; 6 (13): 2242-55 and Chinese patent application CN101490081A (all of which are incorporated herein by reference); or can be obtained by methods known in the art, such as the method described in detail in U.S. patent application US 2008 / 0234183, all of which are incorporated herein by reference.

[0026] The CPP that can be used for the fusion protein of the present invention includes but is not limited to: cationic: Penetratin, HIV-TAT-47-57, HIV-1Rev 34-50, FHV coat-35-49, Oligoarginines (R9-R12), CCMV Gag-7-25, S413-PV, VP22, BP16, DPV3, DPV6, FAH coat protein, protamine 1, human cJun, Engrailed-2, Islet-1, HoxA-13, TP10, etc.; amphipathic: transport peptide (transportan), Transportan 10, Pep-1, MPGα, MPGβ, CADY, Pepfect6, Pepfect14, Pepfect15, NickFect, Hel, sC18, pVEC, ARF (1-22), YTA2, PAR1 (Palmitoyl-SFLLRN), F2Pal10 (Palmitoyl-SFLLRN), BPrPp (1-30), hLF peptide (19-40), Buforin 2, Crotamine, Azurin p18, hCT peptide (18-32), S413-PVrev, etc.; hydrophobic: Kaposi's sarcoma fibroblast growth factor, signal peptide of Ig light chain from Caiiman crocodylus, integrin β3 fragment, Grb2-SH2 domain, HIV-1gp41 (1-23), HBV translocation motif (translocation motif), sperm-egg fusion protein (89–111), human calcitonin (9–32), Pep-7, C105Y, K-FGF, etc.

[0027] In addition, the CPP used in the fusion protein of the present invention can also be selected from a polypeptide sequence having about 60, 70, 80, 90, 95, 99% or 100% sequence identity with any of the polypeptide sequences described above, as long as the polypeptide sequence still retains its biological activity, i.e., promotes cellular uptake of the molecule.

[0028] In certain embodiments, the cell-penetrating peptide is selected from Penetratin (SEQ ID NO: 40), Tat-derived peptide, Rev (34-50) (SEQ ID NO: 42), VP22 (SEQ ID NO: 43), transportan (SEQ ID NO: 44), Pep-1 (SEQ ID NO: 45), Pep-7 (SEQ ID NO: 46), and any combination thereof. In certain embodiments, the Tat-derived peptide is selected from Tat (48-60) (SEQ ID NO: 14) or Tat (47-57) (SEQ ID NO: 41).

[0029] In certain embodiments, the cell-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60). In certain embodiments, the cell-penetrating peptide comprises or consists of the following sequence: SEQ ID NO:14.

[0030] In certain embodiments, the protease is selected from furin and / or lysosomal cysteine ​​protease. In certain embodiments, the protease recognition sequence is selected from furin recognition sequence, lysosomal cysteine ​​protease recognition sequence and a combination thereof.

[0031] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: R-X1-X2-R ↓ (SEQ ID NO: 47), wherein X1 is any amino acid, X2 is K or R, and ↓ indicates a cleavage site.

[0032] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RR-X1-X2-R ↓ (SEQ ID NO:48).

[0033] In certain embodiments, X1 is selected from alanine (A), arginine (R), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), histidine (H), isoleucine (I), glycine (G), aspartic acid (N), leucine (L), lysine (K), methionine (M), phenylalanine (F), coline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0034] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RRHKR ↓ (SEQ ID NO:49).

[0035] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: QSVASSRRHKR ↓ FAGV (SEQ ID NO: 8).

[0036] In certain embodiments, the lysosomal cysteine ​​protease is selected from cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D, or cathepsin H.

[0037] In certain embodiments, the lysosomal cysteine ​​protease is cathepsin L.

[0038] In certain embodiments, the cathepsin L recognition sequence comprises or consists of the following sequence: NNTHDLVGDVRLAGV (SEQ ID NO: 10).

[0039] In certain embodiments, the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence. In certain embodiments, the protease recognition sequence is a single-chain polypeptide comprising a furin recognition sequence and a cathepsin L recognition sequence from N-terminus to C-terminus, or comprising a cathepsin L recognition sequence and a furin recognition sequence from N-terminus to C-terminus.

[0040] In certain embodiments, the protease recognition sequence comprises RRHKR (SEQ ID NO: 49) and NNTHDLVGDVRLAGV (SEQ ID NO: 10). In certain embodiments, the protease recognition sequence comprises SEQ ID NO: 8 and SEQ ID NO: 10.

[0041] In some embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus: the cell-penetrating peptide, the pH-sensitive fusion peptide, the protease recognition sequence, and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion polypeptide, or between any two adjacent domains. In certain exemplary embodiments, the multimerization domain sequence is located at the N-terminus of the fusion polypeptide, or at the C-terminus of the fusion polypeptide.

[0042] In other embodiments, the fusion polypeptide comprises, from N-terminus to C-terminus: the pH-sensitive fusion peptide, the cell-penetrating peptide, the protease recognition sequence, and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion polypeptide, or between any two adjacent domains. In certain exemplary embodiments, the multimerization domain sequence is located at the N-terminus of the fusion polypeptide, or at the C-terminus of the fusion polypeptide.

[0043] In certain embodiments, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus. In certain embodiments, the protease recognition sequence comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

[0044] In certain embodiments, any adjacent domains contained in the fusion polypeptide are optionally connected by a peptide linker. In certain embodiments, the peptide linker is (G m S) n , wherein m is selected from an integer of 1-4 (e.g., 1, 2, 3 or 4), and n is selected from an integer of 1-3 (e.g., 1, 2 or 3). In certain embodiments, the peptide linker is SEQ ID NO: 50. In certain embodiments, the peptide linkers between any adjacent domains may be the same or different.

[0045] In certain exemplary embodiments, the fusion polypeptide comprises the sequence shown in any one of SEQ ID NOs: 16-18.

[0046] In a second aspect, the present invention provides a multimer of the fusion polypeptide described in the first aspect.

[0047] In certain embodiments, the multimer is a dimer, trimer or tetramer. In certain embodiments, the multimer is a dimer.

[0048] In certain embodiments, the multimer is a homomultimer.

[0049] Fusion Protein

[0050] In a third aspect, the present invention provides a fusion protein comprising the fusion polypeptide described in the first aspect and another polypeptide.

[0051] In certain embodiments, the additional polypeptide comprises a detectable label, such as an enzyme, a fluorescent protein, or biotin, among others.

[0052] In certain embodiments, the additional polypeptide comprises an epitope tag, a reporter gene encoding a protein sequence, and / or a nuclear localization signal (NLS) sequence.

[0053] Epitope tags that can be used in the present invention are well known to those skilled in the art, examples of which include but are not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select a suitable epitope tag according to the desired purpose (e.g., purification, detection or tracing). In certain exemplary embodiments, the additional polypeptide comprises a His tag.

[0054] The reporter gene sequences that can be used in the present invention are well known to those skilled in the art, and examples thereof include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0055] The nuclear localization signal (NLS) sequence that can be used in the present invention is well known to those skilled in the art, and examples thereof include but are not limited to the NLS of the SV40 virus large T antigen. In certain exemplary embodiments, the NLS sequence is shown in SEQ ID NO:22.

[0056] In certain exemplary embodiments, the additional polypeptide is a zinc finger protein (eg, ZFP9), or a protein phosphatase (eg, Ppm1b). In certain exemplary embodiments, the zinc finger protein comprises an NLS sequence.

[0057] In certain embodiments, the additional polypeptide is a nucleic acid binding domain sequence.

[0058] In certain embodiments, the nucleic acid binding domain sequence is a zinc finger protein (eg, ZFP9).

[0059] In certain embodiments, the nucleic acid binding domain sequence comprises the sequence shown in SEQ ID NO:31.

[0060] In certain exemplary embodiments, when the additional polypeptide is a zinc finger protein (eg, ZFP9), the multimerization domain sequence comprises the sequence shown in SEQ ID NO:2.

[0061] In certain exemplary embodiments, the fusion protein comprises the sequence shown in any one of SEQ ID NOs: 19-21.

[0062] In certain embodiments, the fusion protein comprises from N-terminus to C-terminus: the cell-penetrating peptide, the pH-sensitive fusion peptide, the protease recognition sequence and another polypeptide, and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion protein, or between any two adjacent domains mentioned above. In certain exemplary embodiments, the multimerization domain sequence is located at the N-terminus of the fusion protein, or at the C-terminus of the protease recognition sequence. Preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

[0063] In certain embodiments, the fusion protein comprises from the N-terminus to the C-terminus: the pH-sensitive fusion peptide, the cell-penetrating peptide, the protease recognition sequence and another polypeptide; and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion protein, or between any two adjacent domains mentioned above. In certain exemplary embodiments, the multimerization domain sequence is located at the N-terminus of the fusion protein, or at the C-terminus of the protease recognition sequence. Preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

[0064] In certain embodiments, the additional polypeptide is fused to the C-terminus of the fusion polypeptide.

[0065] In certain embodiments, the additional polypeptide has a molecular weight of less than 10,000 Da, such as less than 5,000 Da, less than 3,000 Da, or less than 1,000 Da.

[0066] In a fourth aspect, the present invention provides a multimer of the fusion protein described in the third aspect.

[0067] In certain embodiments, the multimer is a dimer, trimer or tetramer. In certain embodiments, the multimer is a dimer.

[0068] In certain embodiments, the multimer is a homomultimer.

[0069] Preparation of polymers

[0070] The fusion polypeptide or fusion protein of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods (recombinant technology), or by chemical synthesis methods (such as Fmoc solid phase method). The fusion protein of the present invention is not limited by its production method.

[0071] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion polypeptide or fusion protein of the present invention.

[0072] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is, for example, a plasmid, a cosmid, a phage, and the like.

[0073] On the other hand, the invention provides a host cell comprising the nucleic acid molecule or vector separated as above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0074] On the other hand, a method for preparing the fusion polypeptide or fusion protein of the present invention is provided, which comprises culturing the host cell as described above under conditions allowing expression of the protein, and recovering the fusion polypeptide or fusion protein from the cultured host cell culture, wherein the fusion polypeptide or fusion protein exists in a multimeric form.

[0075] In another aspect, there is provided a method for preparing the multimer of the present invention, comprising culturing the host cell as described above under conditions allowing expression of the protein, and recovering the multimer from the culture of the cultivated host cell.

[0076] Compound

[0077] In a fifth aspect, the present invention provides a complex comprising the polymer described in the second aspect and a cargo molecule; or, comprising the polymer described in the fourth aspect and a cargo molecule. The cargo molecule may be any molecule.

[0078] In certain embodiments, the cargo molecule is selected from the group consisting of proteins, nucleic acids, carbohydrates, lipids, chemical compounds, and any mixtures thereof.

[0079] In certain embodiments, the molecular weight of the cargo molecule is less than 10,000 Da, such as less than 5,000 Da, less than 3,000 Da, or less than 1,000 Da.

[0080] In certain embodiments, the cargo molecule comprises a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a chemiluminescent substance, or biotin.

[0081] In certain embodiments, the cargo molecule comprises a pharmaceutically active agent.

[0082] Peptide or protein

[0083] In some embodiments, the cargo molecule is a peptide or protein. In certain embodiments, the cargo molecule is fused to a fusion polypeptide or fusion protein constituting the multimer. In certain embodiments, the complex comprises the multimer described in the second aspect.

[0084] In certain embodiments, the complex comprises a single-chain polypeptide, wherein:

[0085] (i) the single-chain polypeptide comprises, from N-terminus to C-terminus: the cell-penetrating peptide, the pH-sensitive fusion peptide, the protease recognition sequence and the cargo molecule; and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion protein, or between any two adjacent domains mentioned above; preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus; or,

[0086] (ii) the fusion protein comprises from N-terminus to C-terminus: the pH-sensitive fusion peptide, the cell-penetrating peptide, the protease recognition sequence and the cargo molecule; and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion protein, or between any two adjacent domains mentioned above; preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus; or,

[0087] (iii) the cargo molecule is fused to the C-terminus of the fusion polypeptide.

[0088] Nucleic Acids

[0089] In other embodiments, the cargo molecule is a nucleic acid. In certain embodiments, the nucleic acid is selected from a DNA molecule, an RNA molecule, an siRNA, an antisense oligonucleotide, a ribozyme, an aptamer, and any combination thereof.

[0090] In certain embodiments, the complex comprises the multimer of the fourth aspect, wherein the fusion protein constituting the multimer comprises a nucleic acid binding domain sequence. In certain embodiments, the nucleic acid binding domain sequence is a zinc finger protein (e.g., ZFP9). In certain embodiments, the nucleic acid binding domain sequence comprises the sequence shown in SEQ ID NO:31. In certain exemplary embodiments, when the additional polypeptide is a zinc finger protein (e.g., ZFP9), the multimerization domain sequence comprises the sequence shown in SEQ ID NO:2.

[0091] Connection

[0092] In certain embodiments, the cargo molecule is fused, chemically coupled, or non-covalently linked to the multimer (or a fusion polypeptide or fusion protein constituting the multimer).

[0093] In some embodiments, the multimer described in the second aspect or the fourth aspect (or the fusion polypeptide or fusion protein constituting the multimer) is fused with a cargo molecule. In certain embodiments, the cargo molecule is a peptide or a protein.

[0094] In some embodiments, the multimer described in the second aspect or the fourth aspect (or the fusion polypeptide or fusion protein constituting the multimer) is chemically coupled to the cargo molecule. The "chemical coupling" refers to the bonding obtained in the chemical reaction between the reactive group contained in the multimer (or the fusion polypeptide or fusion protein constituting the multimer) and the reactive group contained in the cargo molecule, and the two parts are connected by a covalent bond after the chemical reaction. Before the above-mentioned chemical reaction (coupling reaction), the multimer, the cargo molecule or both can be modified in an independent reaction with a linker molecule so that they respectively contain the reactive group required for the chemical coupling. The selection of the linker molecule used to modify the multimer or cargo molecule depends on the coupling strategy used.

[0095] In certain embodiments, the covalent bond is a disulfide bond, a phosphodiester bond, a phosphorothioate bond, an amide bond, an amine bond, a thioether bond, an ether bond, an ester bond, or a carbon-carbon bond.

[0096] In certain embodiments, the cargo molecule is coupled to the C-terminus of the multimer.

[0097] In certain embodiments, the cargo molecule is a nucleic acid.

[0098] In other embodiments, the multimer of the second aspect or the fourth aspect is non-covalently linked to the cargo molecule.

[0099] In certain embodiments, the polymer is conjugated to the cargo molecule via electrostatic interaction.

[0100] In certain embodiments, the cargo molecule is a nucleic acid.

[0101] Preparation of complexes

[0102] In some embodiments, when the complex comprises a chemically coupled polymer and a cargo molecule, the complex of the present invention can be obtained by the following exemplary method: under conditions that allow the reactive groups respectively contained in the polymer and the cargo molecule to undergo a chemical reaction, the polymer and the cargo molecule are mixed so that the two parts are connected by a covalent bond. In certain embodiments, the method further comprises: using a linker molecule to modify the polymer, the cargo molecule, or both so that they respectively contain the reactive groups required for the above-mentioned chemical reaction. In certain embodiments, the cargo molecule is a nucleic acid.

[0103] In other embodiments, when the complex comprises a polymer and a cargo molecule conjugated by electrostatic interaction, the complex of the present invention can be obtained by the following exemplary method: (1) mixing the polymer of the present invention with the cargo molecule to form a mixture; and (2) incubating the mixture to allow the polymer to form a complex with the cargo molecule. In certain embodiments, the cargo molecule is a nucleic acid. In such embodiments, the fusion protein constituting the polymer preferably further comprises a nucleic acid binding domain sequence.

[0104] Uses and methods

[0105] The polymers or complexes of the present invention can significantly improve the endocytic efficiency of cargo molecules, allowing the cargo molecules to be efficiently released from endocytic vesicles, and once the cargo molecules are available in the cytoplasm, they can play any role associated with them. Therefore, the polymers or complexes of the present invention can be used as intracellular delivery agents, and thus further used in research and therapeutic and diagnostic applications.

[0106] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the fusion polypeptide, fusion protein, multimer, complex, isolated nucleic acid molecule, vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0107] In certain embodiments, the pharmaceutical composition comprises the complex described in the fifth aspect.

[0108] In certain embodiments, the cargo molecule included in the complex is a pharmaceutically active agent or a detectable marker. The marker can be used for diagnosis, for studying drug disposition (e.g., absorption, distribution, metabolism, excretion), for studying the efficacy or side effects of treatment or drugs, etc.

[0109] On the other hand, the present invention also relates to the use of the fusion polypeptide, fusion protein, multimer, complex or an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding the fusion polypeptide or fusion protein of the present invention in the preparation of a medicament for treating a disease; wherein the cargo molecule contained in the complex is capable of treating the disease.

[0110] In certain embodiments, the disease is a disease associated with programmed cell necrosis, wherein the cargo molecule comprises protein phosphatase 1B (Ppm1b). In certain embodiments, the disease associated with programmed cell necrosis includes liver injury (e.g., drug-induced liver injury, such as acute liver injury caused by acetaminophen APAP), inflammatory diseases (e.g., systemic inflammatory response SIRS), ischemia-reperfusion injury and / or neurodegenerative diseases. In certain embodiments, the disease is a disease associated with programmed cell necrosis caused by TNF-α, such as systemic inflammatory response SIRS caused by TNF-α.

[0111] The fusion polypeptide, fusion protein, polymer, complex or pharmaceutical composition of the present invention can be in any form known in the medical field, for example, in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use.

[0112] The fusion polypeptide, fusion protein, multimer, complex or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, rectal, parenteral or topical administration.

[0113] An exemplary route of administration is oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, etc. In addition to active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl acetate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and mixtures thereof. In addition to inert diluents, liquid dosage forms for oral administration may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics, etc. Solid dosage forms for oral administration include capsules, tablets, pills, lozenges, powders, granules, etc. In addition to the active ingredients, solid dosage forms may contain pharmaceutically acceptable inert excipients or carriers, for example fillers (such as lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, cross-linked polyvinylpyrrolidone and calcium sulfate); binders (such as carboxymethyl cellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose and acacia); wetting agents (such as cetyl alcohol and glyceryl monostearate); disintegrants (such as agar, calcium carbonate, starch, alginic acid, sodium carboxymethyl cellulose, sodium carboxymethyl starch); lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate); and mixtures thereof.

[0114] The fusion polypeptide, fusion protein, multimer, complex or pharmaceutical composition of the present invention can also be administered via a non-oral route.

[0115] Therefore, another exemplary route of administration is parenteral administration, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection and infusion. The dosage form for parenteral administration can be an injection preparation, including injection solution, sterile powder for injection or concentrated solution for injection. In addition to the active ingredient, the injection dosage form can contain a pharmaceutically acceptable carrier such as sterile water, Ringer's solution and isotonic sodium chloride solution, and suitable additives such as antioxidants, buffers and antibacterial agents can also be added according to the properties of the drug.

[0116] Another exemplary route of administration is topical administration, such as transdermal administration (e.g., administration by a transdermal patch or iontophoresis device), intraocular administration, or intranasal or inhaled administration. Dosage forms for transdermal administration may be topical gels, sprays, ointments, and creams. In addition to the active ingredient, the topical dosage form may contain ingredients that enhance the absorption or penetration of the active ingredient through the skin or other area of ​​action.

[0117] Another exemplary route of administration is rectal administration. The dosage form for rectal administration can be a suppository.

[0118] In addition, other carrier materials and administration methods known in the pharmaceutical field may also be used. The fusion polypeptide, fusion protein, multimer, complex or pharmaceutical composition of the present invention may be prepared by any known pharmaceutical process, such as effective preparation and administration method.

[0119] On the other hand, the invention provides a kind of test kit, it contains the fusion polypeptide of the present invention, fusion protein, polymer, complex, isolated nucleic acid molecule, carrier or host cell.In certain embodiments, the test kit further includes instructions for transfection and / or intracellular delivery (intracellular delivery).In certain embodiments, the test kit is used for transfection and / or intracellular delivery of cargo molecules (for example, nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds and any mixture thereof).In certain embodiments, the cell is a mammalian cell, such as a human cell.

[0120] On the other hand, the present invention further relates to fusion polypeptides, fusion proteins, polymers, complexes, isolated nucleic acid molecules, carriers or host cells of the present invention, used as the purposes of delivery reagents (e.g., transfection reagents or intracellular delivery reagents). In certain embodiments, the delivery reagents are used for intracellular delivery of cargo molecules (e.g., nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds and any mixture thereof). In certain embodiments, the cells are mammalian cells, such as human cells.

[0121] In another aspect, the present invention provides a method for delivering a cargo molecule into a cell, comprising: contacting the cell with the complex described in the fifth aspect, wherein the cargo molecule is a cargo molecule contained in the complex.

[0122] In certain embodiments, contacting the cell with the complex is performed in vivo.

[0123] In certain embodiments, the contacting of the cell with the complex is performed ex vivo.

[0124] In certain embodiments, contacting the cell with the complex is performed in vitro.

[0125] In certain embodiments, the cell is a eukaryotic cell, such as a mammalian cell, such as a human cell.

[0126] Definition of terms

[0127] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operating procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0128] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0129] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0130] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0131] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0132] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug (e.g., inhibitory activity on PSD-95 ubiquitination), including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolyzate), etc.

[0133] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0134] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0135] Advantageous Effects of the Invention

[0136] The multimerized delivery system of the present invention can significantly improve the endocytosis efficiency of cargo molecules and the vesicle release efficiency, thereby significantly improving the cytoplasmic delivery efficiency of cargo molecules, so that cargo molecules can give full play to their corresponding biological functions. In particular, the multimerized delivery system of the present invention can significantly improve serum tolerance, making in vivo delivery possible. Therefore, the delivery system of the present invention provides an effective means for affecting the biological mechanisms and pathways of cells, which can be used in many fields such as research, treatment, and diagnosis, and has broad application prospects and clinical value.

[0137] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 The leucine zipper-based multimerization delivery system-eGFP clone design is shown.

[0139] Figure 2 The effect of the leucine zipper on the protein aggregate state is shown.

[0140] Figure 3 Evaluation of the endocytic efficiency and vesicle escape efficiency of the delivery system after the addition of the leucine domain is shown.

[0141] Figure 4 Evaluation of the endocytosis efficiency of the delivery system protein under serum conditions after the addition of the leucine domain is shown.

[0142] Figure 5 Delivery system-eGFP clone designs based on various multimerization domains are shown.

[0143] Figure 6 Fluorescence microscopy imaging shows the effect of various multimerization domains on endocytosis efficiency.

[0144] Figure 7 Evaluation of the effects of various multimerization domains on endocytic efficiency is shown.

[0145] Figure 8 The TINNeL-eGFP / GFPβ1-10 cloning design is shown.

[0146] Fig. 9 Evaluation of endocytic efficiency of the TINNeL delivery system is shown.

[0147] Fig.10 Evaluation of vesicle escape efficiency of the TINNeL delivery system is shown.

[0148] Fig.11 The delivery efficiency evaluation of TINNeL delivery system under different serum concentration conditions is shown.

[0149] Fig.12 A schematic diagram of the transport mechanism of the polymerized delivery system of the present invention is shown.

[0150] Fig.13 The schematic diagram of the clone construction design of TINNeL-Ppm1b delivery system protein and other control proteins is shown.

[0151] Fig.14 The evaluation of the effect of TINNeL-Ppm1b in inhibiting TNF-α-induced cell necrosis is shown.

[0152] Fig.15 The results show that TINNeL-Ppm1b inhibits TNF-α-induced death in mice.

[0153] Fig.16 The HE staining image of the cecum of mice showing that TINNeL-Ppm1b inhibits TNF-α-induced SIRS.

[0154] Fig.17 It was shown that TINNeL-Ppm1b inhibited TNF-α-induced SIRS cecal injury score in mice.

[0155] Fig.18 Evaluation of changes in ALT / AST levels after TINNeL-Ppm1b treatment of APAP-induced acute liver injury in mice is shown.

[0156] Fig.19 Shown is the evaluation of the survival rate of mice treated with TINNeL-Ppm1b following APAP-induced lethality.

[0157] Fig. 20 The schematic diagram of the clone construction design of TINNeL-ZFP9 delivery system protein and other control proteins is shown.

[0158] Fig.21 A schematic diagram of the eukaryotic expression plasmid structure for ZFP9 delivery is shown.

[0159] Fig. 22 Flow cytometric analysis of the transduction efficiency of TINNeL-ZFP9 with a red fluorescent protein expression plasmid is shown.

[0160] Fig.23 Shown is the flow cytometric analysis of TINNeL-ZFP9 and X-tremeGNEN transduced with red fluorescent protein expression plasmid under serum conditions.

[0161] Fig.24The images show the fluorescence expression of Luciferase plasmid transduced with TINNeL-ZFP9 in mice.

[0162] Fig.25 The fluorescence microscopy results of the delivery system transducing GFPβ1-10 are shown.

[0163] Sequence information

[0164] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0165] Table 1: Description of sequences

[0166]

[0167] DETAILED DESCRIPTION

[0168] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0169] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compendium of Molecular Biology Experiments, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturer. It is understood by those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the invention claimed.

[0170] The sources of the main reagents involved in the following examples are as follows:

[0171] The materials required for cloning construction related reagents are as follows: DNA polymerase (TaKaRa, R040A), DNA recovery kit (TianGen, DP214-03), plasmid mini-extraction kit (TianGen, DP103-03), plasmid large-extraction kit (QIAGEN, 12663), Gibson assembly premix 5 tubes (NEB, E2611L), DNA marker (ThmeroFisher, SM0331), agarose (Biowest, BW-R0100),

[0172] The materials required for protein mass expression are as follows: peptone (BiSIGMA-ALDRICH, T7293-1KG), yeast powder (OXOID, LP0021B), sodium chloride (Xilong Chemical, 10011012AR), IPTG (Inalco, 1758-1400)

[0173] The media required for protein purification are as follows: SP SEPHAROSE FAST FLOW (GE Healthcare, 17-0729-01), NI SEPHAROSE (GE Healthcare, 17-5268-02)

[0174] The reagents required for protein purification and storage are as follows: glycerol / propylene glycol / C3H8O3 (SIGMA-ALDRICH, G5516), KCl (Xilong Chemical, 1002007), Na2HPO4·12H2O (Xilong Chemical, 1001067AR), KH2PO4 (Xilong Chemical, 1002048AR500), imidazole (SIGMA-ALDRICH, V900153), Tris base (Seebio, 183995), glucose (Xilong Chemical, 1064008AR500), BCA protein concentration assay kit (Thermo Scientific, 23227);

[0175] Reagents required for cell culture: FBS (GIBCO, 10099-133), DMEM (GIBCO, 11965092), trypsin (AMRESCO, 0458);

[0176] Reagents required for lentiviral packaging and infection: lentiviral packaging plasmids: pCMV-VSV-G (Addgene, 8454), pRSV-Rev (Addgene, 12253), pMDLg / pRRE (Addgene, 12251); X-tremeGENE transfection reagent (Roche, 06366244001), Puromycin (InvivoGen, ant-pr-5), Blasticidin (InvivoGen, ant-bl-5b), polybrene (Santa Cruz, sc-134220);

[0177] The GFPβ1-10, ZFP9, Ppm1b, dsRed, mCherry, and Histone-H3 related plasmids used in the experiment were synthesized by the company (Sangon Biotech), and the plasmid pCasKP-hph (Addgene, 117232) used to amplify the Cas9 sequence;

[0178] Other reagents: TNF-α (Novoprotein, CF09), PI (ThmeroFisher, P3566)

[0179] Cell lines: HEK-293T (human kidney epithelial cells) and L929 (mouse fibroblasts) were purchased from ATCC.

[0180] Example 1: Establishment and performance evaluation of TL multimerization delivery system

[0181] In this example, a multimerization delivery system was established using a leucine zipper, and the effect of multimerization on endocytosis efficiency was observed by delivering green fluorescent protein eGFP, and the effect of multimerization on vesicle escape efficiency was observed by delivering GFPβ1-10-NLS (referred to as GFPβ1-10) and based on the Split-GFP evaluation method, thereby comprehensively evaluating the effect of multimerization on the delivery efficiency of the delivery system. At the same time, by adding serum (FBS) to the system, the difference in endocytosis efficiency before and after multimerization was observed, thereby evaluating the change in serum tolerance.

[0182] 1.1 Construction of the expression vector of the polymerized delivery system (TAT-Leu Zipper)-cargo molecule complex

[0183] Construct an expression vector containing TAT, Leu-Zipper (leucine zipper), and a cargo molecule of a recombinant protein. The schematic diagram of the structure of the nucleic acid encoding each recombinant protein is shown in Figure 1 As shown, the amino acid sequences of each component from N-terminus to C-terminus are shown in the following table. The construction method is as follows: First, the nucleic acid sequences of TAT, leucine zipper, and cargo molecule (eGFP or GFPβ1-10) in the coding delivery system are obtained by PCR amplification, and the various parts are connected by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET21b(+) are introduced at the 5' end of the fragment by upstream priming, and the BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment by downstream priming. The pET-21b(+) plasmid is treated with double enzyme digestion by NdeI and BamHI. The inserted fragment with overlapping sequences is connected to the restriction vector pET-21b(+) by GIBSON assembly.

[0184] Table 2: Components of the delivery system-cargo molecule complex

[0185]

[0186] 1.2 Expression and purification of polymerized delivery system-cargo molecule complex:

[0187] Transform the expression plasmid described in 1.1 into the expression strain E. coli BL21 (DE3); select a single colony from the transformed plate and inoculate it into 5 ml LB liquid medium containing ampicillin resistance and culture it overnight. Then take 1 ml of the overnight culture and transfer it to 500 ml LB liquid medium containing ampicillin resistance and culture it at 37°C and 180 rpm until the OD of the culture reaches 0. 600 At about 0.6, then add the inducer IPTG to a final concentration of 0.2mM, and induce at 25℃ for 8 hours; after the induction expression is completed, centrifuge at 4℃, 7000g for 10 minutes and collect the bacteria; then resuspend the bacteria with 10ml protein purification equilibrium buffer (PBS, 5% glycerol) and perform ultrasonic disruption. Then centrifuge to obtain the supernatant and load it onto the SP strong cation chromatography column of the protein purification system; then use the protein purification system to elute the target protein with protein elution buffer (PBS+0.2M NaCl~PBS+0.6M NaCl to remove impurities, and collect the eluted product with PBS+1.6M NaCl). The protein concentration can be determined using a spectrophotometer or BCA protein concentration determination kit. Each purified fusion protein is stored at -20℃ after aliquoting.

[0188] 1.3 Evaluation of the effect of leucine zipper on the multimerization of delivery system-cargo molecule complexes

[0189] The TAT-eGFP (T-eGFP) and TAT-Leu-eGFP with leucine zipper (TL-eGFP) obtained in 1.2 above were analyzed for their polymer formation status by non-reducing polyacrylamide gel electrophoresis. Figure 2 As shown, the size of the T-eGFP protein in the monomeric state is about 35kD, while the size of the TL-eGFP protein after the addition of the leucine zipper is at 70kD. This experiment shows that the leucine zipper can cause the delivery system protein to form a dimer.

[0190] 1.4 Evaluation of the endocytosis efficiency of the polymerized delivery system-eGFP complex

[0191] HEK-293T cells were seeded into 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 2*10 6 The cells were rinsed three times with serum-free DMEM medium, and then 5 μM TL-eGFP and T-eGFP were added to the serum-free medium. After incubation for 3 hours, the cells were rinsed three times with pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins adsorbed on the cell membrane that failed to enter the cell, and the cells were collected for flow cytometry analysis. The results are shown in Figure 3 As shown, the intracellular fluorescence intensity of the dimerized protein TL-eGFP is 4-5 times that of the monomer (T-eGFP), proving that the polymerization process improves the endocytosis efficiency of the delivery system.

[0192] 1.5 Evaluation of the vesicle escape efficiency of the polymerized delivery system-GFPβ1-10 complex

[0193] In the Split-GFP system, the 11 β-folds of GFP are split into a large fragment (β1-10) and a small fragment (β11), both of which lose their fluorescence activity, but can spontaneously associate and restore the fluorescence properties of GFP if they meet. Based on this, we constructed HEK293T cells that stably express Histone-β11, and at the same time, GFPβ1-10 with a nuclear import signal (NLS) was fused and expressed as a cargo with the multimerization delivery system to be evaluated, and then transduced the stable cell line. When the delivery system transduces GFPβ1-10, it can only combine with GFPβ11 to generate complete GFP after successfully escaping from the endocytic vesicle and entering the cytoplasm or nucleus. Therefore, the efficiency of endocytic vesicle escape can be evaluated by the proportion of GFP and the relative fluorescence intensity.

[0194] 1.5.1 Construction of HEK293T-GFPβ11 cell line

[0195] (1) Construction of GFPβ11 cell line lentiviral plasmid:

[0196] The coding sequence of Histone-H3 (SEQ ID NO: 27) was obtained by PCR amplification, and the GFPβ11 coding sequence (SEQID NO: 28) was designed directly in the upstream primer because it was shorter. The components were connected by multiple rounds of PCR, and in the last round of PCR, the Hind III restriction site and its overlapping sequence upstream of the Hind III restriction site corresponding to the Lenti vector were introduced at the 5' end of the fragment by the upstream primer, and the BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to the Lenti vector were introduced at the 3' end of the fragment by the downstream primer. The Lenti plasmid was double-digested by Hind III and BamH I. The inserted fragment with overlapping sequences was connected to the Lenti vector after restriction digestion by GIBSON assembly.

[0197] (2) Lentivirus packaging, infection and resistance screening cell lines:

[0198] HEK-293T cells were seeded into 6-well plates and cultured overnight. Before plasmid transfection, the number of cells per well was ensured to be approximately 2*10 7 / ml; before transfection, the cells were replaced with serum-free DMEM medium; 1.5μg Lenti recombinant plasmid, 0.75μg pMDL plasmid, 0.45μg pVSV-G plasmid, 0.3μg pREV (mass ratio of 5:3:2:1) were added to 300μl serum-free DMEM respectively, and after slowly blowing evenly, 9μl (1:3) X-tremeGENE transfection reagent was added and slowly blown evenly, and allowed to stand at room temperature for 15min, and then added dropwise to the cell supernatant. After 8h, it was replaced with DMEM containing 10% FBS for continued culture; after 60h, the culture supernatant was collected for infection.

[0199] HEK-293T cells were seeded into 12-well plates and cultured overnight. Before lentiviral infection, the number of cells per well was approximately 2*10 6 / ml (50% density); discard the original cell culture supernatant, add 300μl lentivirus (moi=3) and 700μl 10% FBS DMEM, add polybrene at a concentration of 10μg / ml, centrifuge the cell plate at 2500rpm for 30min under sterile conditions and continue culturing.

[0200] 48 hours after lentiviral infection, cells were passaged at a ratio of 1 / 3, and puromycin was added at a concentration of 2.5 μg / ml for resistance screening; the positive cells obtained by screening were cloned to obtain a monoclonal cell line of HEK-293T-Hitone-GFPβ11.

[0201] 1.5.2 Detection of vesicle escape efficiency using the Split-GFP system

[0202] The HEK-293T Histone H3-β11 cells (abbreviated as HEK-293Tβ11) were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, the number of cells in each well was ensured to be approximately 2*10 6 About; rinse the cells three times with serum-free DMEM medium, then add 5μM TL-GFPβ1-10 and T-GFPβ1-10 to the serum-free medium, incubate for 3 hours, rinse three times with pre-cooled serum-free DMEM containing 20U / mL heparin to remove proteins adsorbed on the cell membrane that failed to enter the cell, then change to serum DMEM medium and culture for another 9 hours, and collect the cells for flow cytometry analysis. The results showed that the vesicle escape efficiency of the dimerized protein TL-GFPβ1-10 did not change compared with the monomeric T-GFPβ1-10 ( Figure 3 ).

[0203] 1.6 Evaluation of the tolerance of polymer delivery systems to serum conditions

[0204] HEK-293T cells were seeded into 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 2*10 6 About; rinse the cells three times with serum-free DMEM medium, add 5μM TL-eGFP and T-eGFP in serum-free, 5%, 10%, 20%, 50%, 100% FBS DMEM, incubate for 3h, rinse three times with pre-cooled serum-free DMEM containing 20U / mL heparin to remove proteins adsorbed on the cell membrane that failed to enter the cell, then change to serum-free DMEM medium, collect cells for flow cytometry analysis. The results are shown in Figure 4 As shown in the figure, the transduction efficiency of TL-eGFP and T-eGFP decreased with the increase of serum content, but the transduction efficiency of TL-eGFP was relatively less affected by serum. Under 50% FBS conditions, almost no green fluorescence signal was detected in the cells after T-eGFP transduction, while the dimerized protein TL-eGFP still had a transduction efficiency of 70% compared to that without serum. It can be seen that multimerization not only improves the endocytosis efficiency of the protein, but also gives the protein higher serum tolerance.

[0205] 1.7 Effects of different polymerisation domains on the endocytic efficiency of the delivery system

[0206] This example further compares the effects of different multimerization domain sequences on the endocytosis efficiency of the delivery system. The components of the delivery system-cargo molecule complex containing different multimerization domains are shown in the following table. The expression vector expressing the above complex is prepared by the method in 1.1-1.2 above, and the above complexes are expressed and purified. The schematic diagram of the constructed plasmid is shown in Figure 5 shown.

[0207] Table 3: Delivery systems containing different multimerization domains

[0208]

[0209] MA104 cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 2*10 6 The cells were rinsed three times with serum-free DMEM medium, and then 5 μM T-eGFP, TL-eGFP, TL2-eGFP, TN-eGFP, TG-eGFP and TD-eGFP were added to the serum-free medium. After incubation for 3 hours, the cells were rinsed three times with pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins adsorbed on the cell membrane that failed to enter the cell. After replacing the serum-free medium, fluorescence imaging was performed. After the end, the cells were collected for flow cytometry analysis. The fluorescence imaging results are shown in Figure 6As shown in the figure, the intracellular green fluorescence intensity of the T-eGFP group was very low, while the intracellular fluorescence intensity of the other experimental groups with the addition of the multimerization domain increased significantly. Figure 7 As shown, different polymerized domains will enhance the average fluorescence intensity of TAT-mediated eGFP, that is, improve the efficiency of TAT-mediated endocytosis, and the effects of TL-eGFP and TL2-eGFP based on leucine zipper are the most obvious.

[0210] Example 2: Establishment and performance evaluation of TINNeL polymerized delivery system

[0211] In this example, based on the multimeric delivery system of Example 1, a pH-sensitive peptide (INF7) and endocytic vesicle protease-specific cleavage sites (CTSL protease: N, Furin protease: Ne) were further added to construct a TINNEL delivery system, and its endocytic efficiency and vesicle escape efficiency were evaluated.

[0212] 2.1 Construction of TINNEL-cargo complex expression vector

[0213] An expression vector of TINNEL-cargo molecule recombinant protein was constructed. The amino acid sequences of each component contained in each recombinant protein from N-terminus to C-terminus are shown in the following table. The construction method is as follows: First, the nucleic acid sequences encoding TAT, INF7, leucine zipper, N, Ne, and cargo molecules (eGFP or GFPβ1-10) in the delivery system are obtained by PCR amplification, and the various parts are connected by multiple rounds of PCR. In the last round of PCR, the NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) are introduced at the 5' end of the fragment through upstream priming, and the BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment through downstream priming. The pET-21b(+) plasmid is treated with double restriction digestion by NdeI and BamHI. The inserted fragment with overlapping sequences is connected to the restriction vector pET-21b(+) by GIBSON assembly. The schematic diagram of the plasmid is shown in Figure 8 .

[0214] Table 4: Components of the delivery system-cargo molecule complex

[0215]

[0216] 2.2 Expression and purification of TINNeL-cargo molecule complex

[0217] First, the expression plasmid obtained in 2.1 was transformed into the expression strain E.coli BL21 (DE3); a single colony was selected from the transformed plate and inoculated into 5ml LB liquid medium containing ampicillin resistance and cultured overnight, then 1ml of the overnight cultured bacterial solution was transferred to 500mL LB liquid medium containing ampicillin resistance, cultured at 37℃, 180rpm until the bacterial solution OD600 was about 0.6, then the inducer IPTG was added to a final concentration of 0.2mM, and induced at 25℃ for 8 hours; after the induction expression was completed, the bacterial cells were collected after centrifugation at 4℃, 7000g for 10 minutes; then the bacterial cells were resuspended with 10ml protein purification equilibrium buffer (PBS, 5% glycerol) and ultrasonically broken. Then the supernatant was centrifuged and loaded onto the SP strong cation chromatography column of the protein purification system; then the target protein was eluted using the protein purification system with protein elution buffer (PBS+0.2M NaCl~PBS+0.7M NaCl to remove impurities, PBS+1.8M NaCl to collect eluted products). The protein concentration can be measured by spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein is stored at -20℃ after aliquoting.

[0218] 2.3 Evaluation of the intracellular delivery efficiency of the TINNeL delivery system

[0219] Evaluation of endocytosis efficiency: HEK-293T cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 2*10 6 About; rinse the cells three times with serum-free DMEM medium, then add 5 μM of the delivery system protein TINNeL-eGFP and the control protein TINNe-eGFP to the serum-free medium, incubate for 3 hours, rinse three times with pre-cooled serum-free DMEM containing 20 U / mL heparin to remove the proteins adsorbed on the cell membrane that failed to enter the cell, and then collect the cells for flow cytometry analysis. We found that the fluorescence intensity of the dimerized TINNeL-eGFP was 4 times, that is, the endocytosis efficiency was significantly improved after the addition of the leucine zipper domain ( Fig. 9 ).

[0220] Evaluation of vesicle escape efficiency: We used the Split-GFP system to further evaluate the delivery efficiency of the TINNeL system by observing the changes in its escape efficiency. HEK-293Tβ11 was inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, the number of cells in each well was ensured to be approximately 2*10 6The cells were rinsed three times with serum-free DMEM medium, and then 1 μM of the delivery system protein TINNeL-GFPβ1-10 and the control protein TINNe-GFPβ1-10 were added to the serum-free medium. After incubation for 3 hours, the cells were rinsed three times with pre-cooled serum-free DMEM containing 20 U / mL heparin to remove the proteins adsorbed on the cell membrane that failed to enter the cell. After changing to serum medium (10% FBS) and continuing to culture for 9 hours, the cells were collected for flow cytometry analysis. The results are shown in Fig.10 As shown, TINNeL-GFPβ1-10 has a higher fluorescence intensity, indicating that the TINNeL delivery system can simultaneously improve the endocytosis efficiency and vesicle escape efficiency, and is a more efficient delivery method.

[0221] 2.4 Evaluation of the delivery efficiency of TINNeL delivery system under serum conditions

[0222] HEK-293T cells were seeded into 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 2*10 6 About; rinse the cells three times with serum-free DMEM medium, add 5μM TINNeL-GFPβ1-10 and control protein TINNe-GFPβ1-10 in serum-free, 10%, 20%, 50%, 100% FBS DMEM, incubate for 3h, rinse four times with pre-cooled serum-free DMEM containing 20U / mL heparin to remove proteins adsorbed on the cell membrane that failed to enter the cell, then change to serum-free DMEM medium, continue to culture for 9h, and collect cells for flow cytometry analysis. The results are as follows Fig.11 As shown, with the increase of serum content, the fluorescence intensity of TINNeL-GFPβ1-10 and TINNe-GFPβ1-10 groups decreased, but TINNeL-GFPβ1-10 was less affected. Under 100% FBS concentration conditions, almost no green fluorescence signal was detected in the TINNe-GFPβ1-10 group, while TINNeL-GFPβ1-10 still had an efficiency of 70% compared to when no serum was added. Therefore, TINNeL not only has efficient delivery ability, but also its delivery effect will not be greatly affected under the condition of serum presence. Fig.12 A schematic diagram of the TINNeL delivery system is shown.

[0223] In addition, the inventors also evaluated the vesicle escape efficiency of the delivery system containing the protease recognition sequence N (TIN-GFPβ1-10) and the delivery system containing the protease recognition sequence Ne (TINe-GFPβ1-10) based on the Split-GFP system. The difference between the above delivery system and TINNe-GFPβ1-10 is that the protease recognition sequence is replaced by a single N or Ne. The results are as follows Fig.25 As shown, compared with TI-GFPβ1-10 or TIN-GFPβ1-10 without the addition of the protease recognition sequence, the endocytic vesicle escape efficiency was significantly improved whether it was the delivery system containing the single protease recognition sequence N (TIN-GFPβ1-10) or the delivery system containing the protein protease recognition sequence Ne (TINe-GFPβ1-10). The above results indicate that the single protease recognition sequence N or Ne has the ability to improve the vesicle escape efficiency.

[0224] Example 3: Application of TINNeL-Ppm1b in inhibiting TNF-α-induced cell apoptosis

[0225] Cell death can be divided into apoptosis and necrosis. Among them, cell necrosis will cause cell membrane rupture, swelling and leakage of contents, thereby causing a severe inflammatory response, which is mainly controlled by receptor-interaction kinase 3 (RIP3). Under the stimulation of TNF-α, necrosomes containing Rip1 and Rip3 are formed in cells. Rip3 in necrosomes recruits and phosphorylates Mlkl. Phosphorylated Mlk1 translocates to the cell membrane to execute necroptosis. In this process, the phosphorylation of Rip3 is necessary for recruiting Mlk1 to necroptosis factors. Therefore, the phosphorylation process of Rip3 is likely to become an important target for inhibiting cell necrosis and controlling the inflammatory response caused by it. Studies have shown that protein phosphatase 1B (Ppm1b) can inhibit necroptosis in cultured cells and mice by dephosphorylating Rip3. Ppm1b protein has become an important tool for controlling programmed necrosis. Given that it has been found that programmed cell necrosis is closely related to the occurrence of many diseases such as inflammatory diseases, ischemia-reperfusion injury, and neurodegenerative diseases, Ppm1b protein shows great potential in treating the above-mentioned diseases related to programmed cell necrosis.

[0226] 3.1 Construction of expression vectors for TINNeL-Ppm1b complex and other control proteins

[0227] A recombinant protein expression vector containing TAT, INF7, a protease cleavage site, a multimerization domain, and a cargo molecule Ppm1b (SEQ ID NO: 29) was constructed. The structural schematic diagrams of each recombinant protein are shown in FIG. Fig.13As shown, the amino acid sequences of each component from N-terminus to C-terminus are shown in the following table. The construction method is as follows: the nucleic acid sequences of TAT, INF7, N, Ne, and Ppm1b are obtained by PCR amplification, and the various parts are connected by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) are introduced at the 5' end of the fragment by upstream priming, and the BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment by downstream priming. The pET-21b(+) plasmid is treated with double enzyme digestion by NdeI and BamHI. The inserted fragment with overlapping sequences is connected to the restriction vector pET-21b(+) by GIBSON assembly.

[0228] Table 5: Components of the delivery system-cargo molecule complex

[0229]

[0230] 3.2 Expression and purification of TINNeL-Ppm1b protein and other control proteins

[0231] First, the expression plasmid obtained in 3.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was selected from the transformed plate and inoculated into 5 ml LB liquid culture medium containing ampicillin resistance and cultured overnight, then 1 ml of the overnight culture was transferred to 500 ml LB liquid culture medium containing ampicillin resistance, and cultured at 37°C, 180 rpm until the OD600 of the culture was about 0.6, then IPTG was added to induce the final concentration to 0.2 mM, and induced at 25°C for 8 hours; after the induction expression was completed, the cells were collected after centrifugation at 4°C, 7000g for 10 minutes, and some cells were taken to detect the induced expression of the protein; then the cells were resuspended in 10 ml protein purification equilibrium buffer (50 ml C3H8O3, 3.6342 g Tris (Hydroxymethyl) aminomethane dissolved in 1 L double distilled water, adjusted to pH 8.0), and ultrasonically disrupted. Then centrifuge to obtain the supernatant and load it onto the Sulphopropyl (SP) cation exchange column of the AKTA protein purification system; then use the equilibrium buffer and high salt eluent (50ml C3H8O3, 116.88g NaCl, 3.6342g Tris (Hydroxymethyl) aminomethane dissolved in 1L double distilled water, adjust the pH to 8.0) to perform gradient elution in different proportions to obtain the target protein. The protein concentration can be determined using a spectrophotometer or a BCA protein concentration determination kit. Each purified fusion protein is stored at -20°C after aliquoting.

[0232] 3.3 Effect of TINNeL-Ppm1b on TNF-α-induced L292 cell necrosis

[0233] L929 cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 2*10 6 About 10 minutes, rinse the cells three times with serum-free DMEM medium, then add the delivery system protein TINNeL-Ppm1b and other control proteins to the serum-free medium, incubate for 3 hours, add 10% FBS DMEM diluted with 20ng / ml TNF-α and 1ml 20mM z-VAD, incubate for 10 hours, collect the cells for PI staining, and analyze the cells by flow cytometry to observe the rate of cell necrosis. At the same time, use lentivirus to transduce Ppm1b (Lenti-Ppm1b) and lentivirus (Lenti-vec) as a control, package the collected lentivirus with Ppm1b expression sequence and control lentivirus on HEK-293T cells, infect L929 cells for 24 hours, so that Ppm1b is fully expressed in the cells, and the infected L929 cells are replated for TNF-α stimulation. The results are as follows Fig.14 As shown, after TINNeL-Ppm1b treatment, the proportion of cell necrosis caused by TNF-α in L929 cells was the lowest, only about 10%, and its inhibitory effect was even better than that of the lentivirus infection group (20%) as a positive control.

[0234] At the same time, because Ppm1b inhibits cell necrosis by inhibiting the phosphorylation of Rip3, we attempted to verify the content of Rip3 and phosphorylated Rip3 (p-Rip3) in L929 cells of different treatment groups (treatment methods are the same as above) by protein immunoblotting, and further determined that TINNe-Ppm1b inhibits TNF-α cell necrosis by more efficiently inhibiting the phosphorylation of Rip3 after entering the cytoplasm. The results are as follows Fig.14 As shown in the figure, the content of Rip3 in L929 cells of each treatment group was basically the same, while for the phosphorylated p-Rip3, the TINNeL-Ppm1b group was significantly lower than other protein groups. This result indicated that TINNeL-Ppm1b could enter the L929 cytoplasm more efficiently and inhibit the cell necrosis caused by TNF-α by inhibiting the phosphorylation process of Rip3.

[0235] 3.4 Evaluation of the inhibitory activity of TINNeL-Ppm1b on TNF-α-induced systemic inflammatory response syndrome

[0236] In mice, cell necrosis caused by TNF-α further triggers systemic inflammatory response syndrome (SIRS) and eventually leads to death. The TINNeL delivery system not only has a high delivery efficiency, but also should be able to maintain a high delivery effect in the presence of serum, that is, it can still play a good delivery role in the body. Therefore, we tried to observe whether TINNeL-Ppm1b still has a good delivery effect in the body based on the TNF-α-induced SIRS model.

[0237] 25 mmol of TINNeL-Ppm1b and other control proteins (12 mice in each group) were injected into 6-week-old female BALB / C mice through the tail vein. 2 hours later, 15 μg of TNF-α was also injected into the mice through the tail vein. The survival of the mice was observed every hour thereafter. Fig.15 The results showed that during the observation period (survival rate was basically stable after 40 hours), all mice in the control group died within 20 hours due to inflammation caused by TNF-α, while 100% of the mice in the TINNeL-Ppm1b group survived at the end of the observation period ( Fig.15 ); At the same time, SIRS caused by TNF-α can be directly observed from the degree of damage to the cecum of mice. Therefore, we injected 25 nmol of TINNeL-Ppm1b and other control proteins (6 mice in each group) into 6-week-old female BALB / C mice through the tail vein. 2 hours later, 15 μg of TNF-α was also given to the mice through the tail vein. The mice were euthanized 10 hours later, and the cecum was removed for HE staining. The damage to the cecum of mice in different treatment groups was observed and scored. The results showed that the cecum of mice in the TINNeL-Ppm1b group was almost not damaged ( Fig.16 ), the score was comparable to that of the Mock group (the control group that did not receive TNF-α), and it was more effective in inhibiting TNF-α-induced SIRS than other proteins ( Fig.17 ). Therefore, based on the above results, we confirmed that TINNeL-Ppm1b can effectively inhibit TNF-α-induced SIRS in mice, and TINNeL is a more effective delivery system.

[0238] Example 4: Application of TINNeL-Ppm1b in the treatment of APAP-induced acute liver injury

[0239] Acetaminophen (APAP) is a commonly used antipyretic and analgesic drug in clinical practice, but its overdose can cause acute liver damage and pose a risk of death. Recent studies have confirmed that APAP-induced acute liver damage is caused by an inflammatory response through Rip3-mediated programmed cell necrosis. Therefore, dephosphorylating Rip3 by Ppm1b is likely to achieve the effect of treating APAP-induced acute liver damage.

[0240] First, 500 mg / kg (non-lethal dose) of APAP was injected into 5-7 week old mice (16-18 g) through the tail vein. Two injections of 25 nmol of TINNeL-Ppm1b and other control proteins prepared in Example 3 were injected through the tail vein 2h and 6h later (7 mice in each group). Blood was collected from the eye sockets 6h after APAP injection (12h) to detect the levels of ALT and AST in the serum and evaluate the liver damage of the mice. The results showed that the ALT and AST levels in the TINNeL-Ppm1b group were the lowest, which was basically consistent with the APAP dilution buffer DMSO group (the increase in ALT / AST was caused by DMSO, which was not produced through the Rip3 pathway). Therefore, TINNeL-Ppm1b can achieve a relatively ideal therapeutic effect on acute liver damage caused by APAP ( Fig.18 ).

[0241] If the dosage of APAP is further increased, it will cause more serious liver damage and induce the death of mice. Therefore, we tried to observe whether TINNeL-Ppm1b can treat liver damage in mice under the lethal dose of APAP. At the same time, we used NAC, the only drug currently approved by the FDA for the treatment of APAP-induced liver damage, as a control. First, 800 mg / kg (lethal dose) of APAP was injected into 5-7 week old BALB / C mice (16-18g) through the tail vein route, and 25 mmol of TINNeL-Ppm1b and NAC, PBS (7 mice in each group) were injected twice through the tail vein 2h and 6h later, respectively, and the survival of the mice was observed every 1h. The results are as follows Fig.19 As shown in the figure, all mice in the PBS group died within 18 hours. During the 72-hour observation period (within 24 hours is the acute phase), the survival rate of mice in the NAC group was 20%, while that of the TINNeL-Ppm1b group was 60%. Therefore, even for more severe acute liver injury caused by APAP, TINNeL-Ppm1b showed a good therapeutic effect, even slightly better than the current clinical drug NAC. The above results fully demonstrate the advantages of TINNeL delivery in vivo.

[0242] Example 5: Evaluation of nucleic acid delivery efficiency of TINNeL-ZFP9 in vivo and in vitro

[0243] 5.1 Construction of TINNeL-ZFP9 and other control protein expression vectors

[0244] A recombinant protein expression vector containing TAT, INF7, a protease cleavage site, a multimerization domain, and a cargo molecule ZFP9 (SEQ ID NO: 31) was constructed. The structural schematic diagram of each recombinant protein is shown in FIG. Fig. 20 As shown, the amino acid sequences of each component from N-terminus to C-terminus are shown in the following table. The construction method is as follows: nucleic acid sequences encoding TAT, INF7, protease cleavage site, leucine zipper, and ZFP9 are obtained by PCR amplification, and each part is connected by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET21b(+) are introduced at the 5' end of the fragment by upstream priming, and the BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to pET21b(+) are introduced at the 3' end of the fragment by downstream priming. The pET-21b(+) plasmid is treated with double enzyme digestion by NdeI and BamHI. The inserted fragment with overlapping sequences is connected to the restriction vector pET-21b(+) by GIBSON assembly.

[0245] Table 6: Components of the delivery system-cargo molecule complex

[0246]

[0247] 5.2 Expression and purification of TINNeL-ZFP9 and control proteins

[0248] First, the expression plasmid obtained in 5.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was selected from the transformed plate and inoculated into 5 ml LB liquid medium containing ampicillin resistance and cultured overnight, then 1 ml of the overnight culture was transferred to 500 ml LB liquid medium containing ampicillin resistance and cultured at 37°C and 180 rpm until the OD of the culture reached 0. 600At about 0.6, then add induction IPTG to a final concentration of 0.2mM, and induce at 25℃ for 8 hours; after the induction expression is completed, centrifuge at 4℃, 7000g for 10 minutes and collect the bacteria, take part of the bacteria to detect the protein induction expression; then resuspend the bacteria with 10ml protein purification equilibrium buffer (50ml C3H8O3, 3.6342g Tris (Hydroxymethyl) aminomethane dissolved in 1L double distilled water, adjust pH to 8.0), and perform ultrasonic disruption. Then centrifuge to obtain the supernatant, and load it onto the Sulphopropyl (SP) cation exchange column of the AKTA protein purification system; then use the equilibrium buffer and high salt eluent (50ml C3H8O3, 116.88g NaCl, 3.6342g Tris (Hydroxymethyl) aminomethane dissolved in 1L double distilled water, adjust pH to 8.0) to perform gradient elution in different proportions to obtain the target protein. The protein concentration can be measured by spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein is stored at -20℃ after aliquoting.

[0249] 5.3 Construction of eukaryotic expression plasmid containing ZFP9 binding site

[0250] An expression vector (pTT5-tdTomato-6BS plasmid) containing the tdTomato coding sequence and the ZFP9 binding site was constructed. Fig.21 As shown. The tdTomato coding sequence (SEQ ID NO: 33) and the ZFP9 binding site 6*binding sites (6 binding sites) (SEQ ID NO: 34) sequences were obtained by PCR amplification, and the two were connected by two rounds of PCR. In the second round of PCR, the Hind III restriction site and its overlapping sequence upstream of the Hind III restriction site corresponding to the pTT5 vector were introduced at the 5' end of the fragment by upstream priming, and the BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to the pTT5 vector were introduced at the 3' end of the fragment by downstream priming. The pTT5 plasmid was double-digested by Hind III and BamH I. The inserted fragment with overlapping sequences was connected to the pTT5 vector after restriction digestion by GIBSON assembly.

[0251] 5.4 Evaluation of the delivery efficiency of plasmid DNA by TINNeL-ZFP9

[0252] First, HEK-293T cells were seeded into 12-well plates and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 5*10 6About; 5μM delivery system protein or other control protein and 5μg pTT5-tdTomato-6BS plasmid were co-incubated at 37℃ for 30min to fully form a complex; the cells were rinsed three times with serum-free DMEM medium, and then the complex was added and incubated for 3h; the DMEM with 10% FBS was replaced and continued to be cultured, and the expression of red fluorescent protein was analyzed by flow cytometry at 12h, 24h, and 36h after the medium was changed. The results are shown in Fig. 22 As shown, compared with other control proteins, the red fluorescence ratio of the TINNeL-ZFP9 group was the highest at the three detection time points. Therefore, TINNeL-ZFP9 was more effectively delivered into the cell nucleus when combined with pTT5-tdTomato-6BS to achieve the expression of the red fluorescent gene on the plasmid.

[0253] 5.5 Evaluation of the delivery efficiency of TINNeL-ZFP9 under serum conditions

[0254] This example investigates the transfection effect of TINNeL-ZFP9 under serum conditions, using the commercial transfection reagent X-tremeGENE that can still work under serum conditions as a control.

[0255] First, HEK-293T cells were seeded into 12-well plates and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 5*10 6 About; 5μM TINNeL-ZFP9 and 5μg of pTT5-tdTomato-6BS plasmid were co-incubated at 37℃ for 30min to fully form a complex; after adding the complex and incubating for 3h under 100% FBS serum conditions and serum-free conditions, 10% FBSDMEM was replaced for continued culture; in the control X-tremeGENE group, 5μg of pTT5-tdTomato-6BS plasmid was mixed with X-tremeGENE at a ratio of 1μg DNA: 3μl X-tremeGENE, and then allowed to stand at room temperature for 30min, incubated for 8h under 100% FBS serum conditions and serum-free conditions, and then replaced with 10% FBS DMEM for continued culture. For the above treatment groups, flow cytometry analysis of red fluorescent protein expression was performed at 12h, 24h, 36h, 48h, 60h, 72h, and 84h after the medium was changed. The results are shown in Fig.23 As shown in the figure, under serum-free conditions, the proportion of red fluorescent cells in the TINNeL-ZFP9 group increased faster, but after reaching the plateau, the proportion was basically the same as that in the X-tremeGENE group. However, under 100% FBS conditions, at each time point, the proportion of red fluorescent cells had a difference of nearly 10%. TINNeL-ZFP9 showed more efficient transfection ability under serum conditions.

[0256] 5.5 Evaluation of the delivery efficiency of TINNeL-ZFP9 in mice

[0257] The pTT5-Luc-6BS expression plasmid with the Luciferase coding gene (SEQ ID NO: 35) and the ZFP9 binding site (SEQ ID NO: 34) was constructed using the same construction method as described in 5.3 above. 4 nmol of TINNeL-ZFP9 and other control proteins TINNe-ZFP9, T-ZFP9 were mixed with 5 μg of the pTT5-Luc-6BS expression plasmid and injected into the left leg muscle of nude mice 30 minutes later. After 24 hours, the images were taken and the fluorescence intensity was analyzed. The results are shown in Figure 5. Fig.24 As shown, basically no fluorescence was detected in the T-ZFP9 and untreated groups, TINNe-ZFP9 had weak fluorescence, and strong fluorescence was detected in the TINNeL-ZFP9 group. Therefore, it was confirmed at the mouse level that TINNeL-ZFP9 can efficiently bind to and deliver DNA.

[0258] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University Xiamen Wantai Canghai Biotechnology Co., Ltd. <120> Multimeric delivery systems for intracellular delivery of molecules <130> IDC200043 <150> CN201911414575.7 <151> 2019-12-31 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 35 <212> PRT <213> Artificial sequence <220> <223> Leucine zipper-1 <400> 1 Gly Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu Ser 1 5 10 15 Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys Leu Ile 20 25 30 Gly Glu Arg 35 <210> 2 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Leucine Zipper-2 <400> 2 Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile 1 5 10 15 Ala Ala Ile Lys Gln Gly Tyr Gly 20 <210> 3 <211> 40 <212> PRT <213> Artificial sequence <220> <223> NOE multimerization domain <400> 3 Ala Pro Val Pro Val Thr Lys Leu Val Cys Asp Gly Asp Thr Tyr Lys 1 5 10 15 Cys Thr Ala Tyr Leu Asp Tyr Gly Asp Gly Lys Trp Val Ala Gln Trp 20 25 30 Asp Thr Ala Val Phe His Thr Thr 35 40 <210> 4 <211> 33 <212> PRT <213> Artificial sequence <220> <223> GCN4-P1 multimerization domain <400> 4 Arg Met Lys Gln Leu Glu Asp Lys Val Glu Glu Leu Leu Ser Lys Asn 1 5 10 15 Tyr His Leu Glu Asn Glu Val Ala Arg Leu Lys Lys Leu Val Gly Glu 20 25 30 Arg <210> 5 <211> 63 <212> PRT <213> Artificial sequence <220> <223> Delta multimerization domain <400> 5 Leu Ala Ala Leu Thr Thr Arg Val Thr Thr Glu Asn Thr Arg Leu Asp 1 5 10 15 Gly Leu Ile Asn Ser Gly Gln Asn Ser Ile Gly Glu Leu Ser Thr Arg 20 25 30 Leu Ser Asn Val Glu Thr Ser Met Val Thr Thr Ala Gly Arg Gly Leu 35 40 45 Gln Lys Asn Gly Asn Thr Leu Asn Val Ile Val Gly Asn Gly Met 50 55 60 <210> 6 <211> 105 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding leucine zipper-1 <400> 6 ggcgacagaa tgaagcagat cgaagacaag atcgaagaaa tcctgagcaa gatctaccac 60 atcgaaaacg aaatcgccag aatcaagaag ctgatcggcg aaaga 105 <210> 7 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding leucine zipper-2 <400> 7 gaaattgcag caattaaaaa agaaattgca gcaattaaac aggaaattgc agcaattaaa 60 cagggttatg gt 72 <210> 8 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence Ne <400> 8 Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val 1 5 10 15 <210> 9 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Ne <400> 9 cagagcgttg caagcagccg tcgtcataaa cgttttgcag gtgtt 45 <210> 10 <211> 15 <212> PRT <213> Artificial sequence <220> <223> CTSL recognition sequence N <400> 10 Asn Asn Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val 1 5 10 15 <210> 11 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding N <400> 11 aacaacactc atgaccttgt cggtgatgtg agattagccg gagtt 45 <210> 12 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> INF7 <400> 12 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly 20 <210> 13 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding INF7 <400> 13 ggcctgttcg aagcaataga aggtttcata gaaaatggtt gggagggaat gatagacggt 60 tggtacggt 69 <210> 14 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Tat(48-60) <400> 14 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 15 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Tat (48-60) <400> 15 ggccgtaaga agcggagaca gcgacgaaga ccgccgcag 39 <210> 16 <211> 100 <212> PRT <213> Artificial sequence <220> <223> Fusion protein TINL <400> 16 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly 1 5 10 15 Gly Ser Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp 20 25 30 Glu Gly Met Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn 35 40 45 Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly 50 55 60 Gly Ser Gly Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile 65 70 75 80 Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys 85 90 95 Leu Ile Gly Glu 100 <210> 17 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein TINeL <400> 17 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly 1 5 10 15 Gly Ser Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp 20 25 30 Glu Gly Met Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Gln Ser 35 40 45 Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val Gly Gly Gly 50 55 60 Gly Ser Gly Asp Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile 65 70 75 80 Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys 85 90 95 Leu Ile Gly Glu Arg 100 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein TINNeL <400> 18 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly 1 5 10 15 Gly Ser Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp 20 25 30 Glu Gly Met Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn 35 40 45 Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly 50 55 60 Gly Ser Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly 65 70 75 80 Val Gly Gly Gly Gly Ser Gly Asp Arg Met Lys Gln Ile Glu Asp Lys 85 90 95 Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala 100 105 110 Arg Ile Lys Lys Leu Ile Gly Glu Arg 115 120 <210> 19 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein TINL-ZFP9 <400> 19 Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile 1 5 10 15 Ala Ala Ile Lys Gln Gly Tyr Gly Gly Gly Gly Gly Ser Gly Arg Lys 20 25 30 Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly Gly Ser Gly 35 40 45 Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met 50 55 60 Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn Thr His Asp 65 70 75 80 Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly Gly Ser Val 85 90 95 Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn 100 105 110 Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr Gly 115 120 125 Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val Arg 130 135 140 His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro Phe 145 150 155 160 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu Gln 165 170 175 Arg His Leu Lys Thr His Leu Arg Gly Ser 180 185 <210> 20 <211> 186 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein TINeL-ZFP9 <400> 20 Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile 1 5 10 15 Ala Ala Ile Lys Gln Gly Tyr Gly Gly Gly Gly Gly Ser Gly Arg Lys 20 25 30 Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly Gly Ser Gly 35 40 45 Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met 50 55 60 Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Gln Ser Val Ala Ser 65 70 75 80 Ser Arg Arg His Lys Arg Phe Ala Gly Val Gly Gly Gly Gly Ser Val 85 90 95 Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg Asn 100 105 110 Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr Gly 115 120 125 Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val Arg 130 135 140 His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro Phe 145 150 155 160 Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu Gln 165 170 175 Arg His Leu Lys Thr His Leu Arg Gly Ser 180 185 <210> 21 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein TINNeL-ZFP9 <400> 21 Glu Ile Ala Ala Ile Lys Lys Glu Ile Ala Ala Ile Lys Gln Glu Ile 1 5 10 15 Ala Ala Ile Lys Gln Gly Tyr Gly Gly Gly Gly Gly Ser Gly Arg Lys 20 25 30 Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Gly Gly Gly Ser Gly 35 40 45 Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met 50 55 60 Ile Asp Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn Thr His Asp 65 70 75 80 Leu Val Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly Gly Ser Gln 85 90 95 Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val Gly Gly 100 105 110 Gly Gly Ser Val Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile 115 120 125 Cys Met Arg Asn Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg 130 135 140 Thr His Thr Gly Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn 145 150 155 160 Phe Ser Val Arg His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly 165 170 175 Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser 180 185 190 Thr Ser Leu Gln Arg His Leu Lys Thr His Leu Arg Gly Ser 195 200 205 <210> 22 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NLS <400> 22 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 23 <211> 239 <212> PRT <213> Artificial sequence <220> <223> eGFP <400> 23 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 24 <211> 720 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding eGFP <400> 24 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720 <210> 25 <211> 228 <212> PRT <213> Artificial sequence <220> <223> GFPβ1-10-NLS <400> 25 Met Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Ile Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Ser Phe Lys Asp Asp Gly Lys Tyr Lys Thr Arg Ala Val 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Thr Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Thr Val Arg His Asn Val Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Val Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys His His His His His His Pro Lys Lys 210 215 220 Lys Arg Lys Val 225 <210> 26 <211> 687 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding GFPβ1-10-NLS <400> 26 atgggcagca aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 60 ggtgatgtta atgggcacaa attttctgtc agaggagagg gtgaaggtga tgctacaatc 120 ggaaaactca cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 180 cttgtcacta ctctgaccta tggtgttcaa tgcttttccc gttatccgga tcacatgaaa 240 aggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg cactatatct 300 ttcaaagatg acgggaaata caagacgcgt gctgtagtca agtttgaagg tgataccctt 360 gttaatcgta tcgagttaaa gggtactgat tttaaagaag atggaaacat tctcggacac 420 aaactcgagt acaactttaa ctcacacaat gtatacatca cggcagacaa acaaaagaat 480 ggaatcaaag ctaacttcac agttcgccac aacgttgaag atggttccgt tcaactagca 540 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 600 tacctgtcga cacaaactgt cctttcgaaa gatcccaacg aaaagcacca ccaccaccac 660 caccccaaga agaagaggaa ggtgtaa 687 <210> 27 <211> 411 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding Histone-H3 <400> 27 atggctcgca ctaagcaaac tgctcggaag tctactggtg gcaaggcgcc acgcaaacag 60 ttggccacta aggcagcccg caaaagcgct ccggccaccg gcggcgtgaa aaagccccac 120 cgctaccggc cgggcaccgt ggctctgcgc gagatccgcc gttatcagaa gtccactgaa 180 ctgcttattc gtaaactacc tttccagcgc ctggtgcgcg agattgcgca ggactttaaa acagacctgc gtttccagag ctccgctgtg atggctctgc aggaggcgtg cgaggcctac 300 ttggtagggc tatttgagga cactaacctg tgcgccatcc acgccaagcg cgtcactatc 360 atgcccaagg acatccagct cgcccgccgc atccgcggag agaggcgtg a <210> 28 <211> 47 <212> DNA <213> The snowstorm <220> <223> The anti-GFPβ11 antibodies were induced <400> 28 47. cgggaccaca tggtgctgca cgagtacgtg aacgccgccg gcatcac <210> 29 <211> 378 <212> PRT <213> The snowstorm <220> <223> Ppm1b <400> 29 Met Gly Ala Phe Leu Asp Lys Pro Lys Thr Glu Lys His Asn Ala His 1 5 10 15 Gly Ala Gly Asn Gly Leu Arg Tyr Gly Leu Ser Ser Met Gln Gly Trp 20 25 30 Arg Val Glu Met Glu Asp Ala His Thr Ala Val Val Gly Ile Pro His 35 40 45 Gly Leu Asp Asn Trp Ser Phe Phe Ala Val Tyr Asp Gly His Ala Gly 50 55 60 Ser Arg Val Ala Asn Tyr Cys Ser Thr His Leu Leu Glu His Ile Thr 65 70 75 80 Thr Asn Glu Asp Phe Arg Ala Ala Asp Lys Ser Gly Ser Ala Leu Glu 85 90 95 Pro Ser Val Glu Ser Val Lys Thr Gly Ile Arg Thr Gly Phe Leu Lys 100 105 110 Ile Asp Glu Tyr Met Arg Asn Phe Ser Asp Leu Arg Asn Gly Met Asp 115 120 125 Arg Ser Gly Ser Thr Ala Val Gly Val Met Val Ser Pro Thr His Met 130 135 140 Tyr Phe Ile Asn Cys Gly Asp Ser Arg Ala Val Leu Cys Arg Asn Gly 145 150 155 160 Gln Val Cys Phe Ser Thr Gln Asp His Lys Pro Cys Asn Pro Val Glu 165 170 175 Lys Glu Arg Ile Gln Asn Ala Gly Gly Ser Val Met Ile Gln Arg Val 180 185 190 Asn Gly Ser Leu Ala Val Ser Arg Ala Leu Gly Asp Tyr Asp Tyr Lys 195 200 205 Cys Val Asp Gly Lys Gly Pro Thr Glu Gln Leu Val Ser Pro Glu Pro 210 215 220 Glu Val Tyr Glu Ile Val Arg Ala Glu Glu Asp Glu Phe Val Val Leu 225 230 235 240 Ala Cys Asp Gly Ile Trp Asp Val Met Ser Asn Glu Glu Leu Cys Glu 245 250 255 Phe Val Lys Ser Arg Leu Glu Val Ser Asp Asp Leu Glu Asn Val Cys 260 265 270 Asn Trp Val Val Asp Thr Cys Leu His Lys Gly Ser Arg Asp Asn Met 275 280 285 Ser Val Val Leu Val Cys Phe Ser Asn Ala Pro Lys Val Ser Glu Glu 290 295 300 Ala Val Lys Arg Asp Ser Glu Leu Asp Lys His Leu Glu Ser Arg Val 305 310 315 320 Glu Glu Ile Met Gln Lys Ser Gly Glu Glu Gly Met Pro Asp Leu Ala 325 330 335 His Val Met Arg Ile Leu Ser Ala Glu Asn Ile Pro Asn Leu Pro Pro 340 345 350 Gly Gly Gly Leu Ala Gly Lys Arg His Val Ile Glu Ala Val Tyr Ser 355 360 365 Arg Leu Asn Pro His Lys Asp Asn Asp Gly 370 375 <210> 30 <211> 1134 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Ppm1b <400> 30 atgggtgcat ttttggataa acccaaaact gaaaagcaca atgctcacgg tgctgggaat 60 ggtctgcgtt atggcctgag cagtatgcaa ggatggagag tagaaatgga agatgcacac 120 acagctgttg tgggtattcc tcacggcttg gacaactggt cgttttttgc agtttatgac 180 ggtcatgctg gatcccgagt ggcaaattac tgttcaacac atctattaga acacatcacc 240 accaatgaag acttcagggc agctgacaaa tcaggctctg ctctcgagcc ttcagtagaa 300 agtgtgaaga ctggtatcag gactggcttt ttgaaaattg atgaatatat gcgtaacttc 360 tcagacctga ggaacgggat ggacaggagc ggctcgactg cagtgggcgt gatggtctcc 420 cctacacaca tgtacttcat caactgtggt gactctcgag ctgttctgtg taggacgga 480 caggtctgct tttctaccca ggatcacaaa ccttgtaatc cagtggagaa ggagcgcatc caaaacgcag gaggcagtgt gatgatccag cgtgtgaacg gctcactagc agtgtctcgg gctctggggg actatgacta caagtgtgtg gatggcaagg gccctacaga gcagcttgtt 660 tctccagagc ctgaggttta tgagattgtg agagcagaag aggatgagtt tgtcgtcttg 720 gcctgtgatg ggatctggga tgtgatgagc aatgaggagc tctgtgagtt tgttaagtct 780 aggcttgagg tgtcggacga cctggagaat gtgtgcaatt gggtagtgga cacttgttta cataaggga gtcgagata catgagtgtt gtattagttt gcttttcaaa tgcccccaag gtctcagagg aagccgtgaa gagagattca gagttggata agcacttgga atcacgggtt gaagaatca tgcagaagtc tggggagga ggaatgcctg atcttgccca tgtgatgcgc attttgtctg cagaaaatat cccgaattta cctcccgggg gaggtctcgc tggcaagcgc catgttattg aagctgttta tagtagactt aatccacaca aagacaatga tggg 1134 <210> 31 <211> 98 <212> PRT <213> Artificial sequence <220> <223> ZFP9-NLS <400> 31 Val Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg 1 5 10 15 Asn Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr 20 25 30 Gly Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val 35 40 45 Arg His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro 50 55 60 Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu 65 70 75 80 Gln Arg His Leu Lys Thr His Leu Arg Gly Ser Pro Lys Lys Lys Arg 85 90 95 Lys Val <210> 32 <211> 294 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding ZFP9-NLS <400> 32 gtctctagac ccggggagcg ccccttccag tgtcgcattt gcatgcggaa cttttcggat 60 aaaactaaat tgagagttca tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg 120 atctgtatgc gaaatttctc cgttagacat aatttgacta gacatctacg tacgcacacc 180 ggcgagaagc cattccaatg ccgaatatgc atgcgcaact tcagtcaatc tacttctttg 240 caaagacacc taaaaaccca cctgagagga tcccccaaga agaagcgtaa ggtg 294 <210> 33 <211> 1431 <212> DNA <213> Artificial sequence <220> <223> tdTomato coding sequence <400> 33 atggtgagca agggcgagga ggtcatcaaa gagttcatgc gcttcaaggt gcgcatggag 60 ggctccatga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag 120 ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt cgcctgggac 180 atcctgtccc cccagttcat gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc 240 cccgattaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc 300 gaggacggcg gtctggtgac cgtgacccag gactcctccc tgcaggacgg cacgctgatc 360 tacaaggtga agatgcgcgg caccaacttc ccccccgacg gccccgtaat gcagaagaag 420 accatgggct gggaggcctc caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc 480 gagatccacc aggccctgaa gctgaaggac ggcggccact acctggtgga gttcaagacc 540 atctacatgg ccaagaagcc cgtgcaactg cccggctact actacgtgga caccaagctg 600 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg ctccgagggc 660 cgccaccacc tgttcctggg gcatggcacc ggcagcaccg gcagcggcag ctccggcacc 720 gcctcctccg aggacaacaa catggccgtc atcaaagagt tcatgcgctt caaggtgcgc 780 atggagggct ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 840 tacgagggca cccagaccgc caagctgaag gtgaccaagg gcggccccct gcccttcgcc 900 tgggacatcc tgtcccccca gttcatgtac ggctccaagg cgtacgtgaa gcaccccgcc 960 gacatccccg attacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 1020 aacttcgagg acggcggtct ggtgaccgtg acccaggact cctccctgca ggacggcacg 1080 ctgatctaca aggtgaagat gcgcggcacc aacttccccc ccgacggccc cgtaatgcag 1140 aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 1200 aagggcgaga tccaccaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 1260 aagaccatct acatggccaa gaagcccgtg caactgcccg gctactacta cgtggacacc 1320 aagctggaca tcacctccca caacgaggac tacaccatcg tggaacagta cgagcgctcc 1380 gagggccgcc accacctgtt cctgtacggc atggacgagc tgtacaagta a 1431 <210> 34 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> ZFP9 Binding Site <400> 34 tgtagatgga g 11 <210> 35 <211> 1653 <212> DNA <213> Artificial Sequence <220> <223> Luciferase Encoding Gene <400> 35 atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct tgaggatgga 60 <h2 style=";text-align:left;direction:ltr">accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacggga ttaccaggga 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ctcctctgga 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780<h2 style=";text-align:left;direction:ltr"> cgagtcgtct tatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg attgacaaat acgatttatc taatttacac gaattgctt ctggggggcgc acctctttcg aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat gggctcactg agactacatc agctattctg attackacccg agggggatga taaaccggggc 1080. gcggtcggta aagttgttcc attttttga gcgaaggttg tggatctgga taccgggaa acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1260. tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct ttaattaat acaaaggata ccaggtggcc cccgctgaat tggagtcgat attgttacaa caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560 gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620 aaggccaaga agggcggaaa gtccaaattg taa 1653 <210> 36 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Influenza virus HA2 <400> 36 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly 20 <210> 37 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> KALA <400> 37 Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25 30 <210> 38 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> GALA <400> 38 Trp Glu Path Path Leu Path Glu Path Leu Path Glu Path Leu Path Glu His 1 5 10 15 Your Way Glu Way Your Way Glu Way Your Glu Way Your Way Way 20 25 30 <210> 39 <211> 26 <212> PRT <213> artificial sequence <220> <223> Melittin <400> 39 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 In Ser Trp In Lys Arg Lys Arg Gln Gln 20 25 <210> 40 <211> 16 <212> PRT <213> artificial sequence <220> <223> Penetratin <400> 40 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 41 <211> 11 <212> PRT <213> artificial sequence <220> <223> HIV-TAT(47-57) <400> 41 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 42 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HIV-1 Rev(34-50) <400> 42 Lys Gln Ala Ile Pro Val Ala Lys 1 5 <210> 43 <211> 34 <212> PRT <213> Artificial sequence <220> <223> VP22 <400> 43 Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 1 5 10 15 Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro 20 25 30 Val Glu <210> 44 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Transportan <400> 44 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 45 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Pep-1 <220> <221> MOD_RES <222> (1) <223> ACETYLATION <220> <221> MOD_RES <222> (21)..(21) <223> AMIDATION (-NH-CH2-CH2-SH) <400> 45 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 46 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Pep-7 <400> 46 Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Gln Tyr 1 5 10 15 <210> 47 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence-1 <220> <221> MISC_FEATURE <222> (2) <223> X = any amino acid <220> <221> MISC_FEATURE <222> (3) <223> X=K or R <400> 47 Arg Xaa Xaa Arg 1 <210> 48 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence-2 <220> <221> MISC_FEATURE <222> (3) <223> X = any amino acid <220> <221> MISC_FEATURE <222> (4) <223> X=K or R <400> 48 Arg Arg Xaa Xaa Arg 1 5 <210> 49 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence-3 <400> 49 Arg Arg His Lys Arg 1 5 <210> 50 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Peptide linker <400> 50 Gly Gly Gly Gly Ser 1 5

Claims

1. A fusion polypeptide consisting of a multimerization domain sequence, a cell-penetrating peptide, a pH-sensitive peptide, and a protease recognition sequence; wherein: The proteases are furin and cathepsin L; and the fusion polypeptide comprises, from N-terminus to C-terminus: the cell-penetrating peptide, the pH-sensitive peptide, and the protease recognition sequence; and the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion polypeptide.

2. The fusion polypeptide according to claim 1, wherein The multimerization domain is a dimerization domain or a trimerization domain.

3. The fusion polypeptide according to claim 1, wherein The multimerization domain is selected from the group consisting of: leucine zipper, NOE, GCN4-P1, Delta, and any combination thereof.

4. The fusion polypeptide according to claim 1, wherein The multimerization domain is selected from leucine zippers.

5. The fusion polypeptide according to claim 4, wherein The multimerization domain sequence is shown in SEQ ID NO: 1 or 2.

6. The fusion polypeptide according to claim 1, wherein The cell-penetrating peptide is selected from the group consisting of Penetratin, Tat-derived peptide, Rev (34-50), VP22, transportan, Pep-1, Pep-7, and any combination thereof.

7. The fusion polypeptide according to claim 1, wherein The amino acid sequence of the cell-penetrating peptide is shown in any one of SEQ ID NOs: 14, 40-46.

8. The fusion polypeptide according to claim 1, wherein The cell-penetrating peptide is Tat (48-60) or Tat (47-57).

9. The fusion polypeptide according to claim 1, wherein The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO:

14.

10. The fusion polypeptide according to claim 1, wherein The pH sensitive peptide is selected from influenza virus HA2, INF7, KALA, GALA, melittin, and any combination thereof.

11. The fusion polypeptide according to claim 1, wherein The amino acid sequence of the pH sensitive peptide is shown in any one of SEQ ID NOs: 12, 36-39.

12. The fusion polypeptide according to claim 1, wherein The amino acid sequence of the pH sensitive peptide is shown in SEQ ID NO:

12.

13. The fusion polypeptide according to claim 1, wherein The furin recognition sequence comprises R-X1-X2-R (SEQ ID NO: 47), wherein X1 is any amino acid and X2 is K or R.

14. The fusion polypeptide according to claim 1, wherein The furin protease recognition sequence is shown in SEQ ID NO:

8.

15. The fusion polypeptide according to claim 1, wherein The cathepsin L recognition sequence is shown in SEQ ID NO:

10.

16. The fusion polypeptide according to claim 1, wherein The protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence.

17. The fusion polypeptide according to claim 1, wherein The protease recognition sequences are SEQ ID NO: 8 and SEQ ID NO:

10.

18. The fusion polypeptide according to claim 16, wherein The protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

19. The fusion polypeptide according to any one of claims 1 to 18, wherein Any two adjacent domains contained in the fusion polypeptide are connected via a peptide linker.

20. The fusion polypeptide according to claim 19, wherein The peptide linker is (G m S) n , wherein m is selected from an integer of 1-4, and n is selected from an integer of 1-3.

21. The fusion polypeptide according to claim 1, wherein The amino acid sequence of the fusion polypeptide is shown in any one of SEQ ID NOs: 16-18.

22. A multimer, which is a multimer of the fusion polypeptide according to any one of claims 1 to 21.

23. The multimer according to claim 22, wherein The multimer is a dimer or a trimer.

24. The multimer according to claim 22, wherein The multimers are homomultimers.

25. A fusion protein comprising the fusion polypeptide of any one of claims 1 to 21, and another polypeptide, wherein the other polypeptide is a nucleic acid binding domain sequence; wherein: (i) the fusion protein comprises, from the N-terminus to the C-terminus: the cell-penetrating peptide, the pH-sensitive peptide, the protease recognition sequence and another polypeptide; and the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion protein; or, (ii) the additional polypeptide is fused to the C-terminus of the fusion polypeptide.

26. The fusion protein of claim 25, wherein The additional polypeptide comprises a nuclear localization signal (NLS) sequence.

27. The fusion protein according to claim 25 or 26, wherein The nucleic acid binding domain sequence is a zinc finger protein.

28. The fusion protein according to claim 25 or 26, wherein The nucleic acid binding domain sequence is ZFP9.

29. The fusion protein according to claim 25 or 26, wherein The nucleic acid binding domain sequence is shown in SEQ ID NO:

31.

30. The fusion protein of claim 25 or 26, wherein The amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 19-21.

31. A multimer, which is a multimer of the fusion protein according to any one of claims 25 to 30.

32. The multimer of claim 31, wherein The multimer is a dimer or a trimer.

33. The multimer of claim 31, wherein The multimers are homomultimers.

34. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion polypeptide of any one of claims 1-21, or the fusion protein of any one of claims 25-30.

35. A vector comprising the isolated nucleic acid molecule of claim 34.

36. A host cell comprising the isolated nucleic acid molecule of claim 34 or the vector of claim 35, wherein: The host cell is not a plant cell or a mammalian cell.

37. prepare the fusion polypeptide described in any one of claims 1-21, or the method for the fusion rotein described in any one of claims 25-30, it comprises, cultivate the host cell of claim 36 under suitable conditions, and reclaim the fusion polypeptide or fusion rotein from cell culture, wherein the fusion polypeptide or fusion rotein exists in multimeric form.

38. A complex comprising the multimer of any one of claims 22 to 24 or the multimer of any one of claims 31 to 33, and a cargo molecule.

39. The complex of claim 38, wherein The cargo molecule is selected from the group consisting of proteins, nucleic acids, carbohydrates, lipids and any mixture thereof.

40. The complex of claim 38, wherein The cargo molecule is fused, chemically coupled or non-covalently linked to the polymer.

41. The complex of claim 38, wherein The cargo molecule is a peptide or a protein, and the multimer is the multimer according to any one of claims 22 to 24; The cargo molecule is fused to the polymer; wherein the complex comprises a single-chain polypeptide, wherein: (i) the single-chain polypeptide comprises, from N-terminus to C-terminus: the cell-penetrating peptide, the pH-sensitive peptide, the protease recognition sequence and the cargo molecule; and the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion protein; or, (ii) the cargo molecule is fused to the C-terminus of the fusion polypeptide.

42. The complex of claim 38, wherein The cargo molecule is a nucleic acid, and the multimer is a multimer formed by the fusion protein according to any one of claims 25 to 30.

43. The complex of claim 42, wherein The nucleic acid is selected from the group consisting of a DNA molecule, an RNA molecule, an antisense oligonucleotide, a ribozyme, an aptamer, and any combination thereof.

44. The complex of claim 42, wherein The nucleic acid is selected from siRNA.

45. A pharmaceutical composition comprising the complex according to any one of claims 38 to 44, and a pharmaceutically acceptable carrier and / or excipient; in, The cargo molecule comprised by the complex is a pharmaceutically active agent or a detectable label.

46. ​​Use of the complex according to any one of claims 38 to 44 or the pharmaceutical composition according to claim 45 in preparing a drug for treating a disease; wherein: The cargo molecule contained in the complex is capable of treating the disease.

47. The use according to claim 46, wherein The disease is a disease associated with programmed cell necrosis, and the cargo molecule comprises protein phosphatase 1B; the disease associated with programmed cell necrosis includes liver damage, inflammatory diseases, ischemia-reperfusion injury and / or neurodegenerative diseases.

48. A kit comprising the fusion polypeptide of any one of claims 1-21, the multimer of any one of claims 22-24, the fusion protein of any one of claims 25-30, the multimer of any one of claims 31-33, the isolated nucleic acid molecule of claim 34, the vector of claim 35, the host cell of claim 36, or the complex of any one of claims 38-44; The kit further comprises instructions for transfection and / or intracellular delivery.

49. Use of the fusion polypeptide of any one of claims 1-21, the multimer of any one of claims 22-24, the fusion protein of any one of claims 25-30, the multimer of any one of claims 31-33, the isolated nucleic acid molecule of claim 34, the vector of claim 35, the host cell of claim 36, or the complex of any one of claims 38-44 for delivery of agents for non-diagnostic therapeutic purposes.

50. A method for delivering a cargo molecule into a cell for non-diagnostic therapeutic purposes, comprising: The cell is contacted in vitro with the complex of any one of claims 38-44, wherein the cargo molecule is a cargo molecule comprised in the complex.

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