Multimerized delivery systems for intracellular delivery of molecules

Abstract

A multimerized delivery system can be used to deliver cargo molecules into cells. The multimerized delivery system can achieve highly efficient endocytosis of cargo molecules and their release from endocytic vesicles, thereby significantly improving the efficiency of cytoplasmic delivery of cargo molecules. Once the cargo molecules are available in the cytoplasm, they can perform any role associated with them. The multimerized delivery system provides an effective means for influencing cellular biological mechanisms and pathways and can be used in various fields, such as research, therapy, and diagnosis.

Description

[Technical field]

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

[0002] The cell membrane, as a selectively permeable barrier, is important for cell survival and function. Small molecules can pass through the cell membrane either by natural cellular processes or by direct diffusion through lipid bilayers, but in most cases, the effective passage of intracellular cargo, such as active biological macromolecules, through the cytoplasmic membrane remains a major obstacle to cellular transport processes. Therefore, molecular transport tools that can effectively improve the efficiency of transporting intracellular cargo into living cells are of great importance for their application in fields such as biomedicine.

[0003] Currently, using cell membrane penetrating peptides (CPPs) to mediate the entry of biological macromolecules into cells is one of the research hotspots in the field of biomedicine. CPPs are generally polypeptides of 5 to 30 amino acids, which can mediate the penetration of biological macromolecules, including proteins, through the cell membrane and entry into cells by chemical crosslinking, fusion expression, or non-covalent binding. Cell membrane penetrating peptides have been used in various fields of biomedicine due to their advantages in mediating the entry of biological macromolecules into cells, such as low dose, short transport time, controllable dose, simple operation, low immune response, and low side effects.

[0004] Although CPPs have the above-mentioned advantages in transporting biological macromolecules, they have some limitations, mainly including their own low delivery efficiency and a further decrease in delivery efficiency in the presence of serum.

[0005] Previous studies have shown that only 1% of biomacromolecules carried by CPPs can successfully escape from endocytic vesicles, and most of these are eventually targeted to lysosomes for degradation. Therefore, the endosomal escape efficiency of intracellular cargo is also an important limiting factor that determines the overall delivery efficiency for the intracellular delivery process of CPPs. Many studies have been directed to solving the problem of low delivery efficiency of CPPs. The main strategy to improve the endosomal escape efficiency is to disrupt the integrity of the vesicle membrane during the process of maturation and acidification, so that its contents (including the delivered intracellular cargo) can be released into the cytoplasm. Currently, the most effective method is to use pH-sensitive membrane-fusogenic peptides derived from viruses, bacteria, animals, and plants, or humans. pH-sensitive peptides contain a certain proportion of hydrophobic amino acids and undergo dramatic conformational changes at low pH. During the process of maturation and acidification of endocytic vesicles after CPP-mediated endocytosis, when the pH value drops to a critical point, the pH-sensitive peptide coupled to the CPP undergoes a conformational change and binds to the lipid bilayer of the vesicle membrane. As a result, the integrity of the phospholipid bilayer membrane is severely disturbed, small pores are formed therein, or the vesicle membrane is ruptured, and the transported biological macromolecules are finally released into the cytoplasm. However, subsequent studies have revealed that although the endocytic vesicle escape efficiency of the intracellular delivery system of macromolecules using CPPs fused with pH-sensitive peptides was indeed improved, a significant portion of the transported macromolecules still remained in the endocytic vesicles (obvious spot-like distribution could be observed when fluorescent proteins were used as the transported macromolecules).

[0006] Another factor limiting the application of cell membrane-penetrating peptides is serum intolerance: under conditions of high serum concentration, CPPs cannot sufficiently interact with cell membranes due to the electrostatic interaction between the positive charges of CPPs and the negative charges of hemoglobin, resulting in a sharp decline in their delivery efficiency, which also severely limits the wide application of CPPs in vivo.

[0007] In summary, although much research has been done to improve the efficiency of CPP-mediated macromolecular entry into cells, the effect is still unsatisfactory. The efficiency of entry into cells remains the major limiting factor for the widespread application of CPPs in the field of developing biopolymer drugs targeting intracellular targets. At the same time, serum resistance is also an issue that must be resolved to use CPPs in drug development, but there are no research reports on this issue. Summary of the Invention

[0008] After extensive experiments and repeated exploration, the inventors of the present application unexpectedly found that the introduction of a multimerization domain can greatly improve the endocytosis efficiency of cargo molecules and greatly improve serum resistance. Furthermore, the combination of a pH-sensitive peptide and a specific protease recognition sequence can greatly 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 achieves effective cytoplasmic delivery.

[0009] Fusion Polypeptides Thus, in a first aspect, the present invention provides a fusion polypeptide comprising a multimerization domain sequence, a cell membrane-permeable peptide, a pH-sensitive membrane fusogenic peptide, and a protease recognition sequence.

[0010] In the present invention, the term "multimerization domain" refers to any polypeptide or protein capable of multimerizing (i.e. forming a biomolecular complex) several copies of a fusion polypeptide as described herein. In certain embodiments, the multimerization domain is a homomultimerization domain that aggregates identical fusion polypeptides to form multimers.

[0011] 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 interaction between at least two domains (and the polypeptide of which it is a part).

[0012] 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.

[0013] In certain embodiments, the multimerization domain is selected from a leucine zipper.

[0014] In certain embodiments, the multimerization domain sequence comprises the sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0015] In the present invention, the term "pH-sensitive membrane fusogenic peptide" is used interchangeably with "pH-sensitive peptide" and refers to a class of polypeptides that undergo conformational changes under acidic conditions (e.g., pH less than 6.5) and can thereby promote fusion with endocytic vesicle membranes. During the process of maturation and acidification of endocytic vesicles after the pH-sensitive peptide is endocytosed by cells, when the pH value drops to a critical point, such peptides undergo conformational changes and bind to the lipid bilayer of the vesicle membrane, thereby severely disrupting the integrity of the phospholipid bilayer membrane. As a result, the integrity of the phospholipid bilayer membrane is severely disrupted, small pores are formed therein, or the vesicle membrane is ruptured, and the transported biological macromolecules are finally released into the cytoplasm. Such polypeptides are well known in the art and are described, for example, in 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, which are incorporated by reference in their entireties.

[0016] pH-sensitive peptides that may be used in the fusion proteins of the invention may be selected from or derived from the following proteins or polypeptides: Viral protein sources: HA2 (influenza virus) and its variants KALA, GALA, penton base (adenovirus or rhinovirus), gp41 (HIV), L2 (papillomavirus), envelope protein (West Nile virus); Bacterial protein sources: Listeriolysin O (LLO), Pneumococcal pneumolysin (PLO), Streptococcal streptolysin O (SLO), Diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Shiga toxin, Cholera toxin; Plant protein sources: ricin, saporin, gelonin toxins; Human / Animal Protein Source: Human calcitonin, fibroblast growth factor receptor, melittin; Synthetic peptides: (R-Ahx-R) (4) AhxB, penetratin (pAntp), EB1, bovine prion protein (bPrPp), sweet arrow peptide (SAP), poly(L-histidine), proline-rich peptide.

[0017] In certain embodiments, the pH sensitive fusogenic peptide is selected from influenza virus HA2 (SEQ ID NO: 36) or a variant thereof, melittin (SEQ ID NO: 39), and any combination thereof. In certain embodiments, the variant of influenza virus HA2 is selected from INF7 (SEQ ID NO: 12), KALA (SEQ ID NO: 37), or GALA (SEQ ID NO: 38).

[0018] In certain embodiments, the pH sensitive fusogenic peptide comprises INF7. In certain embodiments, the pH sensitive fusogenic peptide comprises or consists of the following sequence: SEQ ID NO: 12.

[0019] In the present invention, the term "cell membrane penetrating peptide (CPP)", also referred to as "cell penetrating peptide", "protein translocation domain (PTD)", "Trojan horse peptide", "transduction peptide", etc., refers to a polypeptide that can promote cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids). Such polypeptides are well known in the art and can be obtained by methods known in the art, such as those described in Stewart KM, et al. Org Biomol Chem. 2008 Jul 7;6(13):2242-55, and China Patent Publication No. 101490081 (all of which are incorporated herein by reference in their entirety), or those described in US Patent Publication No. 2008 / 0234183 (which is incorporated herein by reference in its entirety).

[0020] CPPs that can be used in the fusion protein of the present invention include, but are not limited to, cationic types: penetratin, HIV-TAT-47-57, HIV-1 Rev 34-50, FHV coat-35-49, oligoarginine (R9 to R12), CCMV Gag-7-25, S413-PV, VP22, BP16, DPV3, DPV6, FAH coat, protamine 1, human cJun, engrailed-2, Islet-1, HoxA-13, TP10, etc., amphipathic types: transportan, transportan 10, Pep-1, MPGα, MPGβ, CADY, Pepfect6, Pepfect14, Pepfect15, NickFect, Hel, sC 18, 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 type: Kaposi's sarcoma fibroblast growth factor, Ig light chain signal peptide from Caiiman crocodylus, integrin β3 fragment, Grb2-SH2 domain, HIV-1 gp41(1-23), HBV transfer motif, sperm-egg fusion protein(89-111), human calcitonin(9-32), Pep-7, C105Y, K-FGF, etc.

[0021] Furthermore, the CPP used in the fusion proteins of the present invention may also be selected from polypeptide sequences having about 60%, 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any of the above polypeptide sequences, so long as the polypeptide sequence still retains its biological activity, i.e., promotes cellular uptake of the molecule.

[0022] In certain embodiments, the cell membrane penetrating peptide is selected from the group consisting of 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).

[0023] In certain embodiments, the cell membrane penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60). In certain embodiments, the cell membrane penetrating peptide comprises or consists of the following sequence: SEQ ID NO:14.

[0024] In certain embodiments, the protease is selected from a furin and / or a lysosomal cysteine ​​protease, hi certain embodiments, the protease recognition sequence is selected from the group consisting of a furin recognition sequence, a lysosomal cysteine ​​protease recognition sequence, and combinations thereof.

[0025] 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 the cleavage site).

[0026] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RR-X1-X2-R ↓ (Sequence number 48).

[0027] 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), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0028] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RRHKR ↓ (Sequence number 49).

[0029] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: QSVASSRRHKR ↓ FAGV (sequence number 8).

[0030] In certain embodiments, the lysosomal cysteine ​​protease is selected from the group consisting of cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D, or cathepsin H.

[0031] In certain embodiments, the lysosomal cysteine ​​protease is cathepsin L.

[0032] In certain embodiments, the cathepsin L recognition sequence comprises or consists of the following sequence: NNTHDLVGDVRLAGV (SEQ ID NO: 10).

[0033] 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 polypeptide chain that comprises, from N-terminus to C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or that comprises, from N-terminus to C-terminus, a cathepsin L recognition sequence and a furin recognition sequence.

[0034] In certain embodiments, the protease recognition sequences include RRHKR (SEQ ID NO:49) and NNTHDLVGDVRLAGV (SEQ ID NO:10). In certain embodiments, the protease recognition sequences include SEQ ID NO:8 and SEQ ID NO:10.

[0035] In some embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus, a cell membrane penetrating peptide, a pH-sensitive membrane fusogenic peptide, and a 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 described above. In certain exemplary embodiments, the multimerization domain sequence is at the N-terminus of the fusion polypeptide or at the C-terminus of the fusion polypeptide.

[0036] In other embodiments, the fusion polypeptide comprises, from the N-terminus to the C-terminus, a pH-sensitive membrane fusogenic peptide, a cell membrane penetrating peptide, and a 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 described above. In certain exemplary embodiments, the multimerization domain sequence is at the N-terminus of the fusion polypeptide or at the C-terminus of the fusion polypeptide.

[0037] In certain embodiments, the protease recognition sequence comprises, from N-terminus to C-terminus, a furin recognition sequence and a cathepsin L recognition sequence. In certain embodiments, the protease recognition sequence comprises, from N-terminus to C-terminus, a cathepsin L recognition sequence and a furin recognition sequence.

[0038] In certain embodiments, any adjacent domains included in the fusion polypeptide are optionally linked by a peptide linker. In certain embodiments, the peptide linker is (G m S) nwhere m is selected from an integer from 1 to 4 (e.g., 1, 2, 3, or 4) and n is selected from an integer from 1 to 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.

[0039] In certain exemplary embodiments, the fusion polypeptide comprises a sequence set forth in any one of SEQ ID NO:16-SEQ ID NO:18.

[0040] In a second aspect, the present invention provides a multimer of the fusion polypeptide of the first aspect.

[0041] In certain embodiments, the multimer is a dimer, trimer, or tetramer. In certain embodiments, the multimer is a dimer.

[0042] In certain embodiments, the multimer is a homomultimer.

[0043] Fusion proteins In a third aspect, the present invention provides a fusion protein comprising the fusion polypeptide of the first aspect and a further polypeptide.

[0044] In certain embodiments, the additional polypeptide comprises a detectable label, such as an enzyme, a fluorescent protein, or biotin.

[0045] In certain embodiments, the additional polypeptide comprises an epitope tag, a protein sequence encoded by a reporter gene, and / or a nuclear localization signal (NLS) sequence.

[0046] Epitope tags that may be used in the present invention are well known to those of skill in the art, examples of which include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and the skilled artisan will be familiar with how to select an appropriate epitope tag for a desired purpose (e.g., purification, detection, or tracking). In certain exemplary embodiments, the additional polypeptide comprises a His tag.

[0047] Reporter gene sequences that can be used in the present invention are well known to those skilled in the art, and examples include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, and the like.

[0048] Nuclear localization signal (NLS) sequences that can be used in the present invention are well known to those of skill in the art and include, but are not limited to, the NLS of the large T antigen of the SV40 virus. In certain exemplary embodiments, the NLS sequence is shown in SEQ ID NO:22.

[0049] In certain exemplary embodiments, the additional polypeptide is a zinc finger protein (e.g., ZFP9) or a protein phosphatase (e.g., Ppmlb). In certain exemplary embodiments, the zinc finger protein comprises an NLS sequence.

[0050] In certain embodiments, the additional polypeptide is a nucleic acid binding domain sequence.

[0051] In certain embodiments, the nucleic acid binding domain sequence is a zinc finger protein (eg, ZFP9).

[0052] In certain embodiments, the nucleic acid binding domain sequence comprises the sequence shown in SEQ ID NO:31.

[0053] 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.

[0054] In certain exemplary embodiments, the fusion protein comprises a sequence set forth in any one of SEQ ID NO:19-SEQ ID NO:21.

[0055] In certain embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a cell membrane penetrating peptide, a pH-sensitive membrane fusogenic peptide, a protease recognition sequence, and an additional 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 described above. In certain exemplary embodiments, the multimerization domain sequence is located at the N-terminus of the fusion protein or C-terminal to the protease recognition sequence. Preferably, the protease recognition sequence comprises, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, a cathepsin L recognition sequence and a furin recognition sequence.

[0056] In certain embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a pH-sensitive membrane fusogenic peptide, a cell membrane penetrating peptide, a protease recognition sequence, and an additional 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 described 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, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, a cathepsin L recognition sequence and a furin recognition sequence.

[0057] In certain embodiments, the additional polypeptide is fused to the C-terminus of the fusion polypeptide.

[0058] 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.

[0059] In a fourth aspect, the present invention provides a multimer of the fusion protein of the third aspect.

[0060] In certain embodiments, the multimer is a dimer, trimer, or tetramer. In certain embodiments, the multimer is a dimer.

[0061] In certain embodiments, the multimer is a homomultimer.

[0062] Preparation of multimers 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 techniques) or by chemical synthesis methods (e.g., Fmoc solid phase method). The fusion protein of the present invention is not limited by the manner in which it is produced.

[0063] Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion polypeptide or fusion protein of the invention.

[0064] In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule described above. In certain embodiments, the vector is, for example, a plasmid, cosmid, phage, etc.

[0065] In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above, including, but not limited to, prokaryotic cells, such as E. coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0066] In another aspect, there is provided a method for preparing a fusion polypeptide or fusion protein of the invention, comprising culturing a host cell as described above under conditions allowing expression of the protein and recovering the fusion polypeptide or fusion protein from the culture of the cultured host cells, wherein the fusion polypeptide or fusion protein is present in a multimeric form.

[0067] In another aspect, a method for preparing a multimer of the invention is provided, comprising culturing a host cell as described above under conditions that allow expression of the protein, and recovering the multimer from the culture of the cultured host cells.

[0068] Complex In a fifth aspect, the present invention provides a complex comprising a multimer of the second aspect and a cargo molecule, or comprising a multimer of the fourth aspect and a cargo molecule. The cargo molecule may be any molecule.

[0069] In certain embodiments, the cargo molecule is selected from the group consisting of a protein, a nucleic acid, a carbohydrate, a lipid, a chemical compound, and any mixture thereof.

[0070] In certain embodiments, the cargo molecule 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.

[0071] In certain embodiments, the cargo molecule comprises a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a chemiluminescent substance, or biotin.

[0072] In certain embodiments, the cargo molecule comprises a pharma- ceutical active agent.

[0073] Peptides or proteins 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 that forms a multimer. In certain embodiments, the complex comprises a multimer of the second aspect.

[0074] In certain embodiments, the complex comprises a single chain polypeptide, wherein: (i) the single-chain polypeptide comprises, from the N-terminus to the C-terminus, a cell membrane penetrating peptide, a pH-sensitive membrane fusogenic peptide, a protease recognition sequence, and a 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 described above, and preferably the protease recognition sequence comprises, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or, from the N-terminus to the C-terminus, a cathepsin L recognition sequence and a furin recognition sequence; or (ii) the fusion protein comprises, from the N-terminus to the C-terminus, a pH-sensitive membrane fusogenic peptide, a cell membrane penetrating peptide, a protease recognition sequence, and a 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 described above, and preferably, the protease recognition sequence comprises, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or, from the N-terminus to the C-terminus, a cathepsin L recognition sequence and a furin recognition sequence; or (iii) the cargo molecule is fused to the C-terminus of the fusion polypeptide.

[0075] nucleic acid In other embodiments, the cargo molecule is a nucleic acid. In certain embodiments, the nucleic acid is selected from the group consisting of a DNA molecule, an RNA molecule, an siRNA, an antisense oligonucleotide, a ribozyme, an aptamer, and any combination thereof.

[0076] In certain embodiments, the complex comprises a multimer of the fourth aspect, wherein the fusion proteins constituting the multimer comprise 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 set forth 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 set forth in SEQ ID NO:2.

[0077] Connection Style In certain embodiments, the cargo molecules are fused, chemically coupled, or non-covalently linked to the multimer (or a fusion polypeptide or protein that comprises the multimer).

[0078] In some embodiments, the multimer of the second or fourth aspect (or the fusion polypeptide or fusion protein comprising the multimer) is fused to a cargo molecule. In certain embodiments, the cargo molecule is a peptide or protein.

[0079] In some embodiments, the multimer of the second or fourth aspect (or the fusion polypeptide or fusion protein constituting the multimer) is chemically coupled to a cargo molecule. "Chemical coupling" refers to a bond obtained in a chemical reaction between a reactive group contained in the multimer (or the fusion polypeptide or fusion protein constituting the multimer) and a reactive group contained in the cargo molecule, after which the two parts are covalently linked together. Prior to the above chemical reaction (the coupling reaction), the multimer, the cargo molecule, or both can be modified in a separate reaction with a linker molecule, so that each contains the reactive groups required for chemical coupling. The choice of the linker molecule used to modify the multimer or the cargo molecule depends on the coupling strategy used.

[0080] 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.

[0081] In certain embodiments, the cargo molecule is coupled to the C-terminus of the multimer.

[0082] In certain embodiments, the cargo molecule is a nucleic acid.

[0083] In other embodiments, the multimer of the second or fourth aspect is non-covalently linked to the cargo molecule.

[0084] In certain embodiments, the multimer is electrostatically conjugated to the cargo molecule.

[0085] In certain embodiments, the cargo molecule is a nucleic acid.

[0086] Preparation of complexes In some embodiments, when the complex comprises a chemically coupled multimer and cargo molecule, the complex of the invention can be obtained by the following exemplary method: mixing the multimer and the cargo molecule under conditions that allow a chemical reaction between reactive groups contained separately on the multimer and the cargo molecule, thereby covalently linking the two moieties. In certain embodiments, the method further comprises modifying the multimer, the cargo molecule, or both, with a linker molecule, so that each of them contains the reactive groups required for the above chemical reaction. In certain embodiments, the cargo molecule is a nucleic acid.

[0087] In other embodiments, when the complex comprises a multimer 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 multimer of the present invention with the cargo molecule to form a mixture, and (2) incubating the mixture to allow the multimer and the cargo molecule to form a complex. In certain embodiments, the cargo molecule is a nucleic acid. In such embodiments, the fusion protein constituting the multimer preferably further comprises a nucleic acid binding domain sequence.

[0088] Use and Method The multimers or complexes of the present invention can significantly improve the endocytosis efficiency of the cargo molecule, allowing for the effective release of the cargo molecule from the endocytic vesicle, where the cargo molecule can perform any role associated with it once it is available in the cytoplasm. Thus, the multimers or complexes of the present invention can be used as intracellular delivery agents for further research, as well as for therapeutic and diagnostic applications.

[0089] Thus, in another aspect, the invention provides a pharmaceutical composition comprising a fusion polypeptide, fusion protein, multimer, complex, isolated nucleic acid molecule, vector, or host cell of the invention and a pharma- ceutically acceptable carrier and / or excipient.

[0090] In certain embodiments, the pharmaceutical composition comprises a conjugate of the fifth aspect.

[0091] In certain embodiments, the cargo molecule included in the conjugate is a pharma- ceutically active agent or a detectable label, which may be used for diagnostics, pharmacokinetic (e.g., absorption, distribution, metabolism, excretion) studies, therapeutic or drug efficacy or side effect studies, etc.

[0092] In another aspect, the present invention also relates to the use of an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding a fusion polypeptide, fusion protein, multimer, complex or a fusion polypeptide or fusion protein of the present invention in the manufacture of a medicament for treating a disease, wherein the cargo molecule comprised in the complex is capable of treating the disease. In another aspect, the present invention also relates to a method of treating a disease, comprising administering to a subject in need thereof an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding a fusion polypeptide, fusion protein, multimer, complex or a fusion polypeptide or fusion protein of the present invention, wherein the cargo molecule comprised in the complex is capable of treating the disease.

[0093] In certain embodiments, the disease is a disease associated with programmed necrosis, wherein the cargo molecule comprises protein phosphatase 1B (Ppm1b). In certain embodiments, the disease associated with programmed necrosis comprises liver injury (e.g., drug-induced liver injury, such as acute liver injury caused by acetaminophen (APAP)), inflammatory disease (e.g., systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, and / or neurodegenerative disease. In certain embodiments, the disease is a disease associated with programmed necrosis caused by TNF-α, such as TNF-α-induced SIRS, i.e., systemic inflammatory response syndrome.

[0094] The fusion polypeptides, fusion proteins, multimers, complexes, or pharmaceutical compositions of the invention may be in any form known in the medical art, such as tablets, pills, suspensions, emulsions, liquids, gels, capsules, powders, granules, elixirs, troches, suppositories, injections (including injection solutions and lyophilized powders), inhalants, sprays, etc. The preferred dosage form will depend on the intended mode of administration and therapeutic application.

[0095] The fusion polypeptides, fusion proteins, multimers, complexes, or pharmaceutical compositions of the invention may be administered by any suitable method known in the art, including, but not limited to, oral, rectal, parenteral, or topical administration.

[0096] An exemplary route of administration is oral administration. Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and the like. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethanol, isopropanol, ethyl acetate, benzyl alcohol, benzyl benzoic acid esters, propylene glycol, 1,3-butanediol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid sorbitan esters, and mixtures thereof. In addition to inert diluents, liquid dosage forms for oral administration may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and aromatic agents. Solid dosage forms for oral administration include capsules, tablets, pills, lozenges, powders, granules, etc. In addition to the active ingredient, the solid dosage form may contain pharma- ceutically acceptable inert excipients or carriers, such as fillers (e.g., lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, crospovidone, and calcium sulfate), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic), humectants (e.g., cetyl alcohol, and glyceryl monostearate), disintegrants (e.g., agar, calcium carbonate, starch, alginic acid, sodium carboxymethylcellulose, sodium carboxymethyl starch), lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate), and mixtures thereof.

[0097] The fusion polypeptides, fusion proteins, multimers, complexes, or pharmaceutical compositions of the invention may also be administered by parenteral routes.

[0098] Therefore, another exemplary administration route is parenteral administration, such as subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and infusion. Dosage forms for parenteral administration can be injection preparations, including solutions for injection, sterile powders for injection, or concentrated solutions for injection. In addition to the active ingredient, the dosage forms for injection can include pharma- ceutically acceptable carriers such as sterile water, Ringer's solution, and isotonic sodium chloride solution, as well as suitable additives such as antioxidants, buffers, and bacteriostatic agents.

[0099] Another exemplary route of administration is topical administration, for example, transdermal administration (e.g., via a transdermal patch or iontophoretic device), intraocular administration, or intranasal or inhalation administration. Dosage forms for transdermal administration can be topical gels, sprays, ointments, and creams. In addition to the active ingredient, topical dosage forms can include ingredients that enhance absorption or penetration of the active ingredient through the skin or other areas of action.

[0100] Another exemplary route of administration is rectal administration. The dosage form for rectal administration may be a suppository.

[0101] Additionally, other carrier materials and modes of administration known in the pharmaceutical art may also be used. The fusion polypeptides, fusion proteins, multimers, complexes, or pharmaceutical compositions of the invention may be prepared by any known pharmaceutical technique, including effective methods of formulation and administration.

[0102] In another aspect, the invention provides a kit comprising the fusion polypeptide, fusion protein, multimer, complex, isolated nucleic acid molecule, vector, or host cell of the invention. In certain embodiments, the kit further comprises instructions for transfection and / or intracellular delivery. In certain embodiments, the kit is used for transfection and / or intracellular delivery of cargo molecules (e.g., nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds, and any mixtures thereof). In certain embodiments, the cell is a mammalian cell, such as a human cell.

[0103] In another aspect, the present invention also relates to the use of the fusion polypeptides, fusion proteins, multimers, complexes, isolated nucleic acid molecules, vectors, or host cells of the present invention as 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 mixtures thereof). In certain embodiments, the cells are mammalian cells, such as human cells.

[0104] In another aspect, the present invention provides a method of delivering a cargo molecule into a cell, the method comprising contacting the cell with a complex of the fifth aspect, wherein the cargo molecule is a cargo molecule comprised in the complex.

[0105] In certain embodiments, contacting the cell with the complex occurs in vivo.

[0106] In certain embodiments, contacting the cell with the complex occurs ex vivo.

[0107] In certain embodiments, contacting the cell with the complex is performed in vitro.

[0108] In certain embodiments, the cell is a eukaryotic cell, such as a mammalian cell, for example a human cell.

[0109] Definition of Terms In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operation steps of cell culture, biochemistry, nucleic acid chemistry, immunology laboratory, etc. used herein are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

[0110] As used herein, the term "vector" refers to a nucleic acid delivery carrier into which a polynucleotide can be inserted. If the vector can express a 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 carried by the vector can be 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Vectors may contain various elements that control expression, including, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may also contain an origin of replication.

[0111] 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 E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9 cells, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0112] As used herein, the term "identity" refers to the degree of correspondence between two polypeptides or two nucleic acids. If the two sequences being compared have the same monomeric subunit base or amino acid at a particular site (e.g., two DNA molecules each have an adenine at a particular site or two polypeptides each have a lysine at a particular site), then the two molecules are identical at that site. The percentage of identity between two sequences is a function of the number of identical sites shared by the two sequences, multiplied by 100, relative to the total number of sites being compared. For example, if 6 of 10 sites in two sequences are identical, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 sites are identical). Generally, the comparison of two sequences is performed to obtain the maximum identity. Such alignments can be performed using computer programs such as the Align program (DNAstar, Inc.) based on the method of Needleman et al. (J. Mol. Biol. 48:443-453, 1970). 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)) incorporated into the ALIGN program (version 2.0), using a PAM120 residue weighting table, with a gap length penalty of 12 and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0113] The 20 common amino acids included herein are expressed in a routine manner. See, for example, Immunology-A Synthesis (2nd ed., 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 one-letter and three-letter abbreviations that are well known in the art. For example, alanine can be represented by A or Ala.

[0114] As used herein, the term "pharmaceutical acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, as well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, and the like. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic surfactants, anionic surfactants, or non-ionic 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 salts and gelatin.Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerol), and the like.Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvant (e.g., complete Freund's adjuvant), and the like.Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, trichloro-tert-butanol, phenol, sorbic acid, and the like.The stabilizer has the meaning commonly understood by those skilled in the art, which is capable of stabilizing the desired activity of the active ingredient in the drug (e.g., inhibitory activity of PSD-95 ubiquitination), and includes, but is 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 hydrolysate), and the like.

[0115] As used herein, the term "treatment" refers to a method carried out to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, narrowing of the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, and alleviation of symptoms (partial or complete). Furthermore, "treatment" may also refer to the prolongation of survival time compared to expected survival time (if not undergoing treatment).

[0116] As used herein, the term "subject" refers to a mammal, such as a primate, such as a human.

[0117] Beneficial Effects of the Invention The multimerized delivery system of the present invention can greatly improve the endocytosis efficiency and endocytosis vesicle release efficiency of the cargo molecule, thereby greatly improving the cytoplasmic delivery efficiency of the cargo molecule, so that the cargo molecule can fully exert its corresponding biological function. In particular, the multimerized delivery system of the present invention can greatly improve serum resistance, thereby enabling in vivo delivery. Therefore, the delivery system of the present invention provides an effective means for influencing the biological mechanisms and pathways of cells, and can be used in many fields such as research, therapy, diagnosis, etc., and thus has broad application prospects and clinical value.

[0118] The embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention, rather than limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments. [Brief description of the drawings]

[0119] [Figure 1] FIG. 1 shows the clone design of the leucine zipper-based multimerization delivery system-eGFP. [Diagram 2] FIG. 1 shows the effect of leucine zipper on protein aggregate state. [Diagram 3] FIG. 1 shows evaluation of endocytosis and vesicle escape efficiency of the delivery system following addition of a leucine zipper domain. [Figure 4] FIG. 1 shows evaluation of the endocytic efficiency of the delivery system under serum conditions after addition of a leucine zipper domain. [Diagram 5] FIG. 1 shows the clonal design of multiple multimerization domain based delivery system-eGFP. [Figure 6] FIG. 1 shows fluorescence microscopy imaging of the effect of various multimerization domains on endocytic efficiency. [Figure 7] FIG. 1 shows an evaluation of the effect of different multimerization domains on endocytic efficiency. [Figure 8] FIG. 1 shows the clone design for TINNeL-eGFP / GFPβ1-10. [Figure 9] FIG. 1 shows the evaluation of the endocytosis efficiency of the TINNeL delivery system. [Figure 10] FIG. 13. Evaluation of vesicular escape efficiency of the TINNeL delivery system. [Figure 11] FIG. 1 shows the evaluation of the delivery efficiency of the TINNeL delivery system under various serum concentrations. [Figure 12] FIG. 1 shows a schematic diagram of the transport mechanism of the multimerized delivery system of the present invention. [Figure 13] FIG. 1 shows clone constructions for proteins of the TINNeL-Ppm1b delivery system and other control proteins. [Figure 14] FIG. 1 shows the effect of TINNeL-Ppm1b on inhibiting TNF-α-induced cell necrosis. [Figure 15] FIG. 1 shows mouse survival following inhibition of TNF-α-induced death by TINNeL-Ppm1b. [Figure 16] FIG. 13 shows HE staining of mouse SIRS cecum caused by TNF-α after inhibition by TINNeL-Ppm1b. [Figure 17] FIG. 1 shows the injury scores of mouse SIRS cecum caused by TNF-α after inhibition by TINNeL-Ppm1b. [Figure 18] FIG. 1 shows changes in ALT / AST levels after TINNeL-Ppm1b treatment of APAP-induced acute liver injury in mice. [Figure 19] FIG. 1 shows the survival rate of mice treated with TINNeL-Ppm1b against death caused by APAP. [Figure 20] FIG. 1 shows clone constructions for proteins of the TINNeL-ZFP9 delivery system and other control proteins. [Figure 21] FIG. 1 shows a schematic of the eukaryotic expression plasmid construct for ZFP9 delivery. [Figure 22] FIG. 1 shows flow cytometry analysis of the transduction efficiency of red fluorescent protein expression plasmid by TINNeL-ZFP9. [Figure 23] FIG. 13 shows flow cytometry analysis of red fluorescent protein expression plasmids transduced by TINNeL-ZFP9 and X-tremeGNEN under serum conditions. [Figure 24]FIG. 13 shows images of mice for fluorescent expression of luciferase plasmid transduced with TINNeL-ZFP9. [Diagram 25] FIG. 2: Fluorescence microscopy of GFPβ1-10 transduction by the delivery system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0120] Sequence information The partial sequence information included in the present invention is shown in Table 1 below.

[0121] [Table 1] EXAMPLES

[0122] The invention will now be described with reference to the following examples, which are not intended to limit the invention but are intended to illustrate the invention.

[0123] Unless otherwise specified, the molecular biology experiments and immunoassays in the present invention were basically performed by referring 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., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995. Restriction enzymes were used according to the conditions recommended by the product manufacturers. Those skilled in the art will understand that the examples are merely illustrative of the present invention and are not intended to limit the scope of the invention described in the claims.

[0124] The main sources of reagents included in the following examples are as follows:

[0125] Materials required for the reagents related to clone construction were as follows: DNA polymerase (TaKaRa, R040A), DNA recovery kit (TianGen, DP214-03), plasmid mini kit (TianGen, DP103-03), plasmid maxi kit (QIAGEN, 12663), Gibson assembly master mix (NEB, E2611L), DNA marker (ThmeroFisher, SM0331), agarose (Biowest, BW-R0100).

[0126] The materials required for large-scale protein expression were as follows: peptone (BiSIGMA-ALDRICH, T7293-1KG), yeast powder (OXOID, LP0021B), sodium chloride (Xilong Chemical Industry, 10011012AR), IPTG (Inalco, 1758-1400).

[0127] Media required for protein purification were as follows: SP Sepharose Fast Flow (GE Healthcare, 17-0729-01), NI Sepharose (GE Healthcare, 17-5268-02).

[0128] The reagents required for protein purification and storage were as follows: glycerol / glycerol / C3H8O3 (SIGMA-ALDRICH, G5516), KCl (Xilong Chemical Industry, 1002007), Na2HPO4·12H2O (Xilong Chemical Industry, 1001067AR), KH2PO4 (Xilong Chemical Industry, 1002048AR500), imidazole (SIGMA-ALDRICH, V900153), Tris base (Seebio, 183995), glucose (Xilong Chemical Industry, 1064008AR500), BCA protein concentration assay kit (Thermo Scientific, 23227).

[0129] Reagents required for cell culture: FBS (GIBCO, 10099-133), DMEM (GIBCO, 11965092), Trypsin (AMRESCO, 0458).

[0130] 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).

[0131] The GFPβ1-10, ZFP9, Ppm1b, dsRed, mCherry, and histone H3-related plasmids used in the experiments were synthesized by Sangon Bio, and the plasmid used to amplify the Cas9 sequence was pCasKP-hph (Addgene, 117232).

[0132] Other reagents: TNF-α (Novoprotein, CF09), PI (ThmeroFisher, P3566).

[0133] Cell lines: HEK-293T (human kidney epithelial cells), L929 (mouse fibroblasts) were purchased from ATCC.

[0134] Example 1: Establishment and performance evaluation of TL multimerized delivery system In this example, a multimerized delivery system was established using leucine zipper. The effect of multimerization on endocytosis efficiency was observed by delivering green fluorescent protein eGFP. The effect of multimerization on vesicle escape efficiency was observed by delivering GFPβ1-10-NLS (briefly called GFPβ-10) based on the Split-GFP evaluation method. This provided a comprehensive evaluation of the effect of multimerization on the delivery efficiency of the delivery system. At the same time, serum (FBS) was added to the system to observe the difference in endocytosis efficiency before and after multimerization, and to evaluate the change in serum resistance.

[0135] 1.1 Construction of expression vector for multimerized delivery system (TAT-Leu Zipper)-cargo molecule complex An expression vector for a recombinant protein containing TAT, Leu-Zipper, and a cargo molecule was constructed, and the structure of the nucleic acid encoding each recombinant protein is shown in FIG. 1. The amino acid sequence of each component from the N-terminus to the C-terminus is shown in the following table. The construction method was as follows: first, the nucleic acid sequence encoding TAT, Leu-Zipper, and cargo molecule (eGFP or GFPβ1-10) in the delivery system was obtained by PCR amplification, and all these parts were linked by multiple rounds of PCR, and during the last round of PCR, the NdeI restriction site in pET21b(+) and the overlap upstream of the corresponding NdeI restriction site were introduced to the 5' end of the fragment by the forward primer, and the BamHI restriction site in pET-21b(+) and the overlap downstream of the corresponding BamHI restriction site were introduced to the 3' end of the fragment by the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The overlapping inserts were ligated into the digested vector pET-21b(+) by GIBSON assembly.

[0136] [Table 2]

[0137] 1.2 Expression and purification of multimerized delivery system-cargo molecule complexes: Transform the expression plasmid described in 1.1 into the expression strain E. coli BL21(DE3), pick a single colony from the transformed plate, inoculate it into 5 ml of LB liquid medium containing ampicillin resistance, and grow it overnight, then take 1 ml of the overnight cultured bacterial liquid and transfer it to 500 ml of LB liquid medium containing ampicillin resistance, and when the bacterial liquid reaches an OD of about 0.6, 600 The cells were cultured at 37°C and 180 rpm until the expression was complete, then the inducer IPTG was added to a final concentration of 0.2 mM and induced at 25°C for 8 hours, and after the end of the induced expression, the cells were harvested after centrifugation at 7000g for 10 minutes at 4°C, then the cells were resuspended in 10 ml of protein purification equilibration buffer (PBS, 5% glycerol) and disrupted by sonication. The supernatant was then collected by centrifugation and loaded onto a SP strong cation chromatography column of a protein purification system, and then the target protein was eluted with a protein elution buffer using the protein purification system (PBS+0.2M NaCl to PBS+0.6M NaCl to remove impurity proteins, and elution products were collected with PBS+1.6M NaCl). The protein concentration can be determined according to a spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at -20°C.

[0138] 1.3 Evaluation of the effect of leucine zipper multimerization on delivery system-cargo complexes The aggregate formation state of TAT-eGFP (T-eGFP) and TAT-Leu-eGFP (TL-eGFP) containing leucine zipper obtained in 1.2 above was analyzed by non-reducing polyacrylamide gel electrophoresis. The results are shown in Figure 2. The size of T-eGFP protein in the monomer state was about 35 kD, while the protein size of TL-eGFP after adding leucine zipper was 70 kD. This experiment showed that the leucine zipper can promote the formation of dimers of the delivery system protein.

[0139] 1.4 Evaluation of endocytic efficiency of multimerized delivery system-eGFP complexes HEK-293T cells were seeded in 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 2 × 10 6 To ensure that the cells were homogenous, the cells were rinsed three times with serum-free DMEM medium, and then TL-eGFP and T-eGFP were added to serum-free medium at 5 μM each. After incubation for 3 h, the cells were rinsed three times with pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins that were adsorbed to the cell membrane and could not enter the cells. The cells were then collected for flow cytometry analysis. The results are shown in Figure 3. The intracellular fluorescence intensity of the dimerized protein TL-eGFP was 4-5 times that of the monomer (T-eGFP), proving that the multimerization process improves the endocytosis efficiency of the delivery system.

[0140] 1.5 Evaluation of vesicle escape efficiency of multimerized delivery system-GFPβ1-10 complex In the Split-GFP system, the 11 β-sheets of GFP were split into a large fragment (β1-10) and a small fragment (β11). Although they both lost their fluorescent activity, they could spontaneously associate when they met and restore the GFP fluorescent property. Based on this, HEK293T cells capable of stably expressing histone-β11 were constructed, and GFPβ1-10 bearing a nuclear entry signal (NLS) was used as a cargo to be fused and expressed with the multimerized delivery system to be evaluated, and then subjected to transduction of stable cell lines. When GFPβ1-10 was transduced by the delivery system, it could bind to GFPβ11 and generate complete GFP only after it successfully escaped from the endocytic vesicle and entered the cytoplasm or nucleus, so that the escape efficiency of the endocytic vesicle could be evaluated by the percentage of GFP and the relative fluorescence intensity ratio.

[0141] 1.5.1 Construction of HEK293T-GFPβ11 cell line (1) Construction of lentiviral plasmid for GFPβ11 cell line: The coding sequence of histone-H3 (SEQ ID NO:27) was amplified by PCR. The coding sequence of GFPβ11 (SEQ ID NO:28) was short and designed directly in the upstream primer. The components were ligated by multiple rounds of PCR, and in the last round of PCR, a HindIII restriction site in the lentivector and an overlap upstream of the corresponding HindIII restriction site were introduced at the 5' end of the fragment by the forward primer, and a BamHI restriction site in the lentivector and an overlap downstream of the corresponding BamHI restriction site were introduced at the 3' end of the fragment by the reverse primer. The lentive plasmid was subjected to double digestion with HindIII and BamHI. The insert fragment with overlap was ligated into the digested lentivector by GIBSON assembly.

[0142] (2) Lentivirus packaging, infection, and cell line resistance screening: HEK-293T cells were seeded in 6-well plates and cultured overnight. Before plasmid transfection, the number of cells per well was approximately 2 × 10 7 Before transfection, cells were transferred to serum-free DMEM medium to ensure that the number of cells was 100 / ml, and 1.5 μg of lenti recombinant plasmid, 0.75 μg of pMDL plasmid, 0.45 μg of pVSV-G plasmid, and 0.3 μg of pREV (mass ratio of 5:3:2:1) were added to 300 μl of serum-free DMEM, followed by slow spraying. 9 μl (1:3) of X-tremeGENE transfection reagent was added, slowly sprayed, left at room temperature for 15 minutes, and added dropwise to the cell supernatant. After 8 hours, the medium was changed to DMEM containing 10% FBS to continue the culture, and after 60 hours, the cell culture supernatant was collected for later infection.

[0143] HEK-293T cells were seeded in 12-well plates and cultured overnight. Before lentivirus infection, the number of cells in each well was approximately 2 × 10 6The original cell culture supernatant was discarded, 300 μl of lentivirus (moi=3) and 700 μl of DMEM with 10% FBS were added, polybrene was added at a concentration of 10 μg / ml, the cell plate was centrifuged at 2500 rpm under sterile conditions for 30 minutes, and then the culture was continued.

[0144] After 48 hours of lentivirus infection, the cells were passaged at a 1 / 3 ratio, 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 the HEK-293T-Hitone-GFPβ11 monoclonal cell line.

[0145] 1.5.2 Detection of vesicle escape efficiency using the split-GFP system The above HEK-293T Histone H3-β11 cells (abbreviated as HEK-293Tβ11) were seeded into 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 2 × 10 6 After ensuring that the cells were 100% pure, the cells were rinsed three times using serum-free DMEM medium, and then TL-GFPβ1-10 and T-GFPβ1-10 were added to serum-free medium at 5 μM each, incubated for 3 hours, and then rinsed three times using pre-cooled serum-free DMEM containing 20 U / ml heparin to remove proteins that could not enter the cells by adsorbing to the cell membrane, and then the medium was changed to serum-containing DMEM medium and cultured for another 9 hours. The cells were then collected and subjected to flow cytometry analysis. The results showed that the vesicle escape efficiency of the dimerized protein TL-GFPβ1-10 was unchanged compared to the monomeric T-GFPβ1-10 (Figure 3).

[0146] 1.6 Evaluation of the resistance of the multimerized delivery system to serum conditions HEK-293T cells were seeded in 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 2 × 10 6To ensure that the cells were 100% FBS, the cells were rinsed three times with serum-free DMEM medium, and then TL-eGFP and T-eGFP were added at 5 μM each to serum-free, 5% FBS, 10% FBS, 20% FBS, 50% FBS, and 100% FBS DMEM medium. After incubation for 3 hours, the cells were rinsed using pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins that could not enter the cells by adsorbing to the cell membrane, and then the medium was replaced with serum-free DMEM medium, and the cells were collected and subjected to flow cytometry analysis. The results are shown in Figure 4. With the increase in serum content, the transduction efficiency of TL-eGFP and T-eGFP decreased, but the transduction efficiency of TL-eGFP was relatively less affected by serum. Under the condition of 50% FBS, the intracellular green fluorescent signal could hardly be detected after the cells were transduced with T-eGFP, but the dimerized protein TL-eGFP still had a transduction efficiency of 70% compared to the case without serum. It could be seen that multimerization not only improves the endocytosis efficiency of the protein but also confers higher serum resistance to the protein.

[0147] 1.7 Effect of different multimerization domains on the endocytic efficiency of the delivery system In this example, the effect of various multimerization domain sequences on the endocytosis efficiency of the delivery system was compared. The components of the delivery system-cargo molecule complexes containing various multimerization domains are shown in the table below. Expression vectors expressing the above-mentioned complexes were prepared by the methods described in 1.1-1.2 above, and the above-mentioned complexes were expressed and purified. A schematic diagram of the constructed plasmids is shown in FIG. 5.

[0148] [Table 3]

[0149] MA104 cells were seeded in 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 2 × 10 6After ensuring that the cells were 100% pure, the cells were rinsed three times with serum-free DMEM medium, and then T-eGFP, TL-eGFP, TL2-eGFP, TN-eGFP, TG-eGFP, and TD-eGFP were added to the serum-free medium at 5 μM each, and 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 that were adsorbed to the cell membrane but could not enter the cells, and the medium was replaced with serum-free medium, after which fluorescence imaging was performed, and the cells were collected and subjected to flow cytometry analysis. The results of fluorescence imaging are shown in Figure 6. The intracellular green fluorescence intensity of the T-eGFP group was very low, while the intracellular fluorescence intensity of the other experimental groups to which the multimerization domain was added increased significantly. The results of flow cytometry analysis are shown in Figure 7. Various multimerization domains were all able to enhance the average fluorescence intensity of eGFP mediated by TAT, that is, to improve the efficiency of TAT-mediated endocytosis, and TL-eGFP and TL2-eGFP based on leucine zipper had the most obvious effect.

[0150] Example 2: Establishment and performance evaluation of TINNeL multimerized delivery system In this example, based on the multimerized delivery system of Example 1, a pH-sensitive peptide (INF7) and an endocytic vesicle protease-specific cleavage site (CTSL protease: N, furin protease: Ne) were further added to construct a TINNEL delivery system, and its endocytosis efficiency and vesicle escape efficiency were evaluated.

[0151] 2.1 Construction of expression vectors for TINNEL-cargo molecule complexes The expression vectors of the recombinant proteins of TINNEL-cargo molecules were constructed, and the amino acid sequences of each component contained in each recombinant protein from the N-terminus to the C-terminus are shown in the following table. The construction method was as follows: first, the nucleic acid sequences encoding TAT, INF7, leucine zipper, N, Ne, and cargo molecule (eGFP or GFPβ1-10) in the delivery system were obtained by PCR amplification, and these parts were ligated by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site in pET-21b(+) and the overlap upstream of the corresponding NdeI restriction site were introduced to the 5' end of the fragment by the forward primer, and the overlap downstream of the BamHI restriction site in pET-21b(+) and the overlap downstream of the corresponding BamHI restriction site were introduced to the 3' end of the fragment by the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragment with the overlap was ligated to the digested vector pET-21b(+) by GIBSON assembly. A schematic diagram of the plasmid is shown in FIG.

[0152] [Table 4]

[0153] 2.2 Expression and purification of TINNeL-cargo complexes: First, the expression plasmid obtained in 2.1 was transformed into the expression strain E. coli BL21 (DE3), a single colony was picked from the transformed plate, inoculated into 5 ml of LB liquid medium containing ampicillin resistance, and cultured overnight, and then 1 ml of the overnight cultured bacterial solution was transferred to 500 mL of LB liquid medium containing ampicillin resistance, and the bacterial solution was cultured until it reached an OD of about 0.6. 600The cells were cultured at 37°C and 180 rpm until the expression was complete, then the inducer IPTG was added to reach a final concentration of 0.2 mM, and induced at 25°C for 8 hours. After the end of induced expression, the cells were collected by centrifugation at 7000g for 10 minutes at 4°C, and then the cells were resuspended in 10 ml of protein purification equilibration buffer (PBS, 5% glycerol) and disrupted by sonication. The supernatant was then collected by centrifugation and loaded onto a SP strong cation chromatography column of a protein purification system, and then the target protein was eluted with a protein elution buffer using the protein purification system (PBS+0.2M NaCl to PBS+0.7M NaCl to remove impurity proteins, and elution products were collected with PBS+1.8M NaCl). The protein concentration can be determined according to a spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at -20°C.

[0154] 2.3 Evaluation of intracellular delivery efficiency of the TINNeL delivery system Assessment of endocytosis efficiency: HEK-293T cells were seeded in 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells per well was approximately 2 × 10 6 To ensure that the cells were homogenous, the cells were rinsed three times with serum-free DMEM medium, and then the delivery system protein TINNeL-eGFP and the control protein TINNe-eGFP were added to the serum-free medium at 5 μM each, and incubated for 3 hours. After rinsing three times using pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins that were adsorbed to the cell membrane but could not enter the cells, the cells were collected and subjected to flow cytometry analysis. It was found that the fluorescence intensity of TINNeL-eGFP after dimerization was four times higher, that is, the endocytosis efficiency was significantly improved after adding the leucine zipper domain (Figure 9).

[0155] Evaluation of vesicle escape efficiency: Using the Split-GFP system, the delivery efficiency of the TINNeL system was further evaluated by observing the changes in its escape efficiency. HEK-293Tβ11 were seeded into 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 2 × 10 6 To ensure that the cells were 100% pure, the cells were rinsed three times with serum-free DMEM medium, and then the protein of the delivery system TINNeL-GFPβ1-10 and the control protein TINNe-GFPβ1-10 were added to the serum-free medium at 1 μM each, and after incubation for 3 hours, the cells were rinsed three times using pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins that were adsorbed to the cell membrane but could not enter the cells. After changing the medium to a serum-containing medium (10% FBS), the culture was continued for another 9 hours, and the cells were collected for flow cytometry analysis. The results were shown in Figure 10, where TINNeL-GFPβ1-10 had higher fluorescence intensity, indicating that the TINNeL delivery system could improve both endocytosis efficiency and vesicle escape efficiency, and was a more effective delivery method.

[0156] 2.4 Evaluation of delivery efficiency of the TINNeL delivery system under serum conditions HEK-293T cells were seeded into 12-well cell culture plates and cultured overnight, so that the number of cells in each well was approximately 2 × 10 before protein treatment. 6After ensuring that the cells were 100% FBS, the cells were rinsed three times with serum-free DMEM medium, and TINNeL-GFPβ1-10 and the control protein TINNe-GFPβ1-10 were added at 5 μM to serum-free, 10% FBS, 20% FBS, 50% FBS, and 100% FBS DMEM medium, respectively, and incubated for 3 hours, after which the cells were rinsed four times using pre-cooled serum-free DMEM containing 20 U / mL heparin to remove proteins that were adsorbed to the cell membrane but could not enter the cells. After replacing the medium with serum-free DMEM medium, the culture was continued for another 9 hours, and then the cells were collected and subjected to flow cytometry analysis. The results are shown in Figure 11. With increasing serum content, the fluorescence intensity of the TINNeL-GFPβ1-10 group and the TINNe-GFPβ1-10 group decreased, but TINNeL-GFPβ1-10 was less affected. Under the condition of 100% FBS concentration, the green fluorescent signal of the TINNe-GFPβ1-10 group was barely detectable, but TINNeL-GFPβ1-10 still had 70% efficiency compared to the serum-free case. Not only does TINNeL have a highly efficient delivery ability, but its delivery efficiency was not significantly affected in the presence of serum. Figure 12 shows a schematic diagram of the principle of the TINNeL delivery system.

[0157] In addition, we 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 above delivery systems differed from TINNe-GFPβ1-10 only in that the protease recognition sequence was replaced by N or Ne alone. The results were shown in Figure 25. Compared with TI-GFPβ1-10 or TIN-GFPβ1-10 without the protease recognition sequence, both the delivery system containing the protease recognition sequence N alone (TIN-GFPβ1-10) and the delivery system containing the protease recognition sequence Ne (TINe-GFPβ1-10) significantly improved the escape efficiency of endocytic vesicles. The above results showed that either the protease recognition sequence N or Ne alone had the ability to improve the vesicle escape efficiency.

[0158] Example 3: Application of TINNeL-Ppm1b in inhibiting apoptosis induced by TNF-α Cell death can be classified into apoptosis and necrosis. Among them, cell necrosis can result in cell membrane rupture, swelling, and leakage of contents, which triggers a severe inflammatory response that is mainly controlled by receptor interacting kinase 3 (RIP3). Under stimulation of TNF-α, necrosomes containing Rip1 and Rip3 are formed in cells, and Rip3 in the necrosome recruits and phosphorylates Mlk1. Phosphorylated Mlk1 translocates to the cell membrane and initiates necroptosis, during which phosphorylation of Rip3 is required for recruitment of Mlk1 to necroptosis. Thus, phosphorylation of Rip3 may be 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, making the Ppm1b protein an important tool for controlling programmed necrosis. Considering that programmed cell necrosis has been found to be closely associated with the development of inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, and other diseases, the Ppm1b protein shows great potential in the treatment of the above-mentioned diseases associated with programmed cell necrosis.

[0159] 3.1 Construction of expression vectors for TINNeL-Ppm1b complex and other control proteins An expression vector was constructed for a recombinant protein containing TAT, INF7, a protease cleavage site, a multimerization domain, and a cargo molecule Ppm1b (SEQ ID NO: 29). A schematic structure of each recombinant protein is shown in FIG. 13. The amino acid sequence of each component from the N-terminus to the C-terminus is shown in the following table. The construction method was as follows: the nucleic acid sequences of TAT, INF7, N, Ne, and Ppm1b were obtained by PCR amplification, and these parts were connected by multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site in pET-21b(+) and the overlap upstream of the corresponding NdeI restriction site were introduced at the 5' end of the fragment by the forward primer, and the BamHI restriction site in pET-21b(+) and the overlap downstream of the corresponding BamHI restriction site were introduced at the 3' end of the fragment by the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The overlapping inserts were ligated into the digested vector pET-21b(+) by GIBSON assembly.

[0160] [Table 5]

[0161] 3.2 Expression and purification of TINNeL-Ppm1b protein and other control proteins First, the expression plasmid obtained in 3.1 was transformed into the expression strain E. coli BL21 (DE3), a single colony was picked from the transformed plate, inoculated into 5 ml of LB liquid medium containing ampicillin resistance, and cultured overnight. 1 ml of the bacterial liquid cultured overnight was taken and transferred to 500 ml of LB liquid medium containing ampicillin resistance, and the bacterial liquid reached an OD of about 0.6. 600The cells were cultured at 37°C and 180 rpm until the expression level reached 1.0, then inducer IPTG was added to a final concentration of 0.2 mM and induced at 25°C for 8 hours. After the induced expression was completed, the cells were harvested by centrifugation at 7000 g for 10 minutes at 4°C, and a portion of the cells was taken to detect the induced expression of the protein. The cells were then resuspended in 10 ml of protein purification equilibration buffer (50 ml of C3H8O3, 3.6342 g of tris(hydroxymethyl)aminomethane was dissolved in 1 L of double distilled water and adjusted to pH 8.0) and disrupted by sonication. The supernatant was then collected by centrifugation and loaded onto a sulfopropyl (SP) cation exchange column of AKTA protein purification system, followed by gradient elution using equilibration buffer and high salt eluent (50ml C3H8O3, 116.88g NaCl, 3.6342g tris(hydroxymethyl)aminomethane dissolved in 1L double distilled water and adjusted to pH 8.0) in different ratios to obtain the target protein. The protein concentration could be determined by spectrophotometer or according to BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at -20℃.

[0162] 3.3 Effect of TINNeL-Ppm1b on TNF-α-induced necrosis of L292 cells L929 cells were seeded in 12-well cell culture plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 2 × 10 6After ensuring that the cells were 100% FBS, the cells were rinsed three times with serum-free DMEM medium, and then the protein of the delivery system TINNeL-Ppm1b and other control proteins were added in serum-free medium, respectively. After 3 hours of incubation, 1 ml of 20 ng / ml TNF-α and 20 mM z-VAD diluted in DMEM with 10% FBS were added. After 10 hours of incubation, the cells were harvested and PI stained. The proportion of cell necrosis in the cells was observed by flow cytometry analysis. At the same time, Ppm1b introduced into lentivirus (Lenti-Ppm1b) and lentivirus (Lenti-vec) were used as controls, and lentiviruses with Ppm1b expression sequences and control lentiviruses were packaged and harvested in HEK-293T cells, and L929 cells were infected for 24 hours to fully express Ppm1b in the cells, after which the infected L929 cells were reseeded and subsequently stimulated with TNF-α. The results are shown in Figure 14. In L929 treated with TINNeL-Ppm1b, the rate of cell necrosis induced by TNF-α was the lowest, at only about 10%, and the inhibitory effect was even superior to that of the lentivirus-infected group as a positive control (20%).

[0163] At the same time, since Ppm1b inhibited cell necrosis by inhibiting the process of Rip3 phosphorylation, we tried to confirm the content of Rip3 and phosphorylated Rip3 (p-Rip3) in L929 cells of various treatment groups (by the same treatment method as above) by Western blot to further determine that TINNe-Ppm1b inhibited TNF-α cell necrosis by more effectively inhibiting the phosphorylation of Rip3 after entering the cytoplasm. The results were shown in Figure 14. Here, the content of Rip3 in L929 cells of all treatment groups was basically the same, but for p-Rip3 in the phosphorylated state, the TINNeL-Ppm1b group showed significantly lower results than other protein groups, indicating that TINNeL-Ppm1b could more effectively enter L929 cytoplasm and inhibit necrosis caused by TNF-α by inhibiting the phosphorylation of Rip3.

[0164] 3.4 Evaluation of the inhibitory activity of TINNeL-Ppm1b against TNF-α-induced systemic inflammatory response syndrome In mice, TNF-α-induced cell necrosis further triggers systemic inflammatory response syndrome (SIRS) in mice, ultimately leading to the death of the mice. The TINNeL delivery system should not only have high delivery efficiency, but also be able to maintain high delivery efficiency in the presence of serum, that is, it could still fully exert its delivery efficiency in vivo. Therefore, based on the TNF-α-induced SIRS model, we tried to observe whether TINNeL-Ppm1b still has ideal delivery efficiency in vivo.

[0165] 25mmol of TINNeL-Ppm1b and other control proteins were injected into 6-week-old female BALB / C mice (12 per group) via the tail vein route, and 2 hours later, 15μg of TNF-α was also injected into the mice via the tail vein route, and the survival of the mice was observed every hour thereafter (Figure 15). The results showed that during the observation period (survival rate was essentially stable after 40 hours), all mice in the control group died from inflammation caused by TNF-α within 20 hours, while the mice in the TINNeL-Ppm1b group were still 100% alive at the end of the observation period (Figure 15). At the same time, the TNF-α-induced SIRS in mice could be directly observed from the degree of cecal damage, so 25 nmol of TINNeL-Ppm1b and other control proteins were injected into 6-week-old female BALB / C mice (6 per group) via the tail vein route, and 2 hours later, 15 μg of TNF-α was also administered to the mice via the tail vein, and 10 hours later, the mice were euthanized, and the cecum was removed and HE stained to observe the cecal damage of the mice in various treatment groups and score the damage. The results showed that the mice in the TINNeL-Ppm1b group had almost intact cecum (Figure 16), and the score was equivalent to that of the mock group (the control group that did not receive TNF-α), and TNF-α-induced SIRS could be inhibited more effectively compared with other proteins (Figure 17). Therefore, based on the above results, it was confirmed that TINNeL-Ppm1b could effectively inhibit TNF-α-induced SIRS in mice, and TINNeL was a more effective delivery system.

[0166] Example 4: Application of TINNeL-Ppm1b in the treatment of acute liver injury caused by APAP Acetaminophen (APAP) is a commonly used antipyretic and analgesic in clinical practice, but its overdose may lead to the risk of acute liver injury and death. In recent years, studies have confirmed that acute liver injury induced by APAP is caused by an inflammatory response induced by programmed cell necrosis mediated by Rip3. Therefore, dephosphorylation of Rip3 by Ppm1b is very likely to achieve the effect of treating acute liver injury induced by APAP.

[0167] First, 5-7 week old mice (16g-18g) were injected with 500mg / kg (non-lethal dose) of APAP via tail vein route, and 2 injections of 25nmol of TINNeL-Ppm1b and other control proteins prepared in Example 3 (7 mice in each group) were injected via tail vein route 2 hours and 6 hours later, respectively, and 6 hours after APAP injection, blood samples were collected by orbital blood sampling, and the levels of ALT and AST in serum were detected to evaluate the liver damage of mice. The results showed that the ALT and AST levels of the TINNeL-Ppm1b group were the lowest, and they were basically the same as those of the APAP dilution buffer DMSO group (the increase in ALT / AST was caused by DMSO, and this part was not brought about by the Rip3 pathway). Therefore, TINNeL-Ppm1b could achieve the ideal therapeutic effect of acute liver damage induced by APAP (Figure 18).

[0168] Further increasing the dose of APAP could lead to more severe liver damage and subsequently induce the death of mice. Therefore, we attempted to observe whether TINNeL-Ppm1b could treat liver damage in mice with a lethal dose of APAP. Meanwhile, NAC, which is the only drug currently approved by the FDA for the treatment of APAP-induced liver damage, was used as a control. First, 800 mg / kg (lethal dose) of APAP was injected into 5-7 week-old BALB / C mice (16 g-18 g) via the tail vein route, and then two injections of 25 mmol of TINNeL-Ppm1b, NAC, and PBS (7 mice in each group) were injected via the tail vein route 2 and 6 hours later, respectively, and the survival of the mice was observed every hour. The results were shown in Figure 19. All mice in the PBS group died within 18 hours. During the 72-hour observation period (the acute phase occurred within 24 hours), the survival rate of mice in the NAC group was 20%, while that in the TINNeL-Ppm1b group could reach 60%. Thus, even for the more severe acute liver injury caused by APAP, TINNeL-Ppm1b showed excellent therapeutic effects, which were even better than the current clinical drug NAC. The above results fully support the advantages of TINNeL in in vivo delivery.

[0169] Example 5: Evaluation of nucleic acid delivery efficiency of TINNeL-ZFP9 in vitro and in vivo 5.1 Construction of expression vectors for TINNeL-ZFP9 and other control proteins An expression vector for a recombinant protein containing TAT, INF7, a protease cleavage site, a multimerization domain, and a cargo molecule ZFP9 (SEQ ID NO: 31) was constructed. The structure of each recombinant protein is shown in FIG. 20, and the amino acid sequence of each component from the N-terminus to the C-terminus is shown in the following table. The construction method was as follows: the nucleic acid sequences encoding TAT, INF7, a protease cleavage site, a leucine zipper, and ZFP9 were obtained by PCR amplification, and these parts were ligated by multiple PCRs, and in the last round of the PCR process, the NdeI restriction site in pET-21b(+) and the overlap upstream of the corresponding NdeI restriction site were introduced at the 5' end of the fragment by the forward primer, and the BamHI restriction site in pET-21b(+) and the overlap downstream of the corresponding BamHI restriction site were introduced at the 3' end of the fragment by the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The overlapping inserts were ligated into the digested vector pET-21b(+) by GIBSON assembly.

[0170] [Table 6]

[0171] 5.2 Expression and purification of TINNeL-ZFP9 and control proteins First, the expression plasmid obtained in 5.1 was transformed into the expression strain E. coli BL21 (DE3), a single colony was picked from the transformed plate, inoculated into 5 ml of LB liquid medium containing ampicillin resistance, and cultured overnight, then 1 ml of the overnight cultured bacterial liquid was taken and transferred to 500 ml of LB liquid medium containing ampicillin resistance, and the bacterial liquid reached an OD of about 0.6. 600The cells were cultured at 37°C and 180 rpm until the expression level reached 1.0, then inducer IPTG was added to a final concentration of 0.2 mM and induced at 25°C for 8 hours. After the induced expression was completed, the cells were harvested by centrifugation at 7000 g for 10 minutes at 4°C, and a portion of the cells was taken to detect the induced expression of the protein. The cells were then resuspended in 10 ml of protein purification equilibration buffer (50 ml of C3H8O3, 3.6342 g of tris(hydroxymethyl)aminomethane was dissolved in 1 L of double distilled water and adjusted to pH 8.0) and disrupted by sonication. The supernatant was then collected by centrifugation and loaded onto a sulfopropyl (SP) cation exchange column of AKTA protein purification system, followed by gradient elution using equilibration buffer and high salt eluent (50ml C3H8O3, 116.88g NaCl, 3.6342g tris(hydroxymethyl)aminomethane dissolved in 1L double distilled water and adjusted to pH 8.0) in different ratios to obtain the target protein. The protein concentration could be determined by spectrophotometer or according to BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at -20℃.

[0172] 5.3 Construction of eukaryotic expression plasmids containing ZFP9 binding sites An expression vector (pTT5-tdTomato-6BS plasmid) containing the tdTomato coding sequence and the ZFP9 binding site was constructed, and its schematic structure is shown in FIG. 21. *The sequence of the nucleotide ...

[0173] 5.4 Evaluation of the delivery efficiency of TINNeL-ZFP9 in plasmid DNA delivery First, HEK-293T cells were seeded in 12-well plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 5 × 10 6 100% , 5μM of the protein of the delivery system or other control protein and 5g of pTT5-tdTomato-6BS plasmid were co-cultured at 37℃ for 30 minutes to completely form the complex, the cells were rinsed with serum-free DMEM medium three times, then the complex was added and incubated for 3 hours, the medium was replaced with DMEM containing 10% FBS, the culture was continued, and after the medium was replaced, the expression of the red fluorescent protein was observed by flow cytometry analysis for 12 hours, 24 hours, and 36 hours, respectively. The results are shown in Figure 22. 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 effective in delivering pTT5-tdTomato-6BS attached to it into the nucleus, and as a result, the expression of the red fluorescent gene on the plasmid was realized.

[0174] 5.5 Evaluation of delivery efficiency of TINNeL-ZFP9 under serum conditions In this example, the transfection effect of TINNeL-ZFP9 under serum conditions was investigated, and the commercially available transfection reagent X-tremeGENE, which can function under serum conditions, was used as a control.

[0175] First, HEK-293T cells were seeded in 12-well plates and cultured overnight. Before protein treatment, the number of cells in each well was approximately 5 × 10 6 To ensure that the number of cells was 1, 5 μM TINNeL-ZFP9 and 5 μg of pTT5-tdTomato-6BS plasmid were co-cultured at 37°C for 30 minutes to allow complete complex formation, the complex was added under serum-free conditions with 100% FBS and incubated for 3 hours, the medium was replaced with DMEM with 10% FBS, and the culture was continued. In the control X-tremeGENE group, 5 μg of pTT5-tdTomato-6BS plasmid and X-tremeGENE were mixed at a ratio of 1 μg DNA:3 μl of X-tremeGENE, then left to stand at room temperature for 30 minutes, and incubated under serum-free conditions with 100% FBS for 8 hours, and then the medium was replaced with DMEM with 10% FBS, and the culture was continued. For the above treatment groups, the expression of red fluorescent protein was observed by flow cytometry analysis for 12, 24, 36, 48, 60, 72, and 84 hours after changing the medium. The results are shown in Figure 23. Under serum-free conditions, the percentage of red fluorescent cells in the TINNeL-ZFP9 group increased more quickly, but after reaching a plateau stage, the percentage was basically the same as that in the X-tremeGENE group. However, under 100% FBS conditions, there was a nearly 10% difference in the percentage of red fluorescent cells at each time point, indicating that TINNeL-ZFP9 showed more effective transfection ability under serum conditions.

[0176] 5.5 Evaluation of delivery efficiency of TINNeL-ZFP9 in mice A pTT5-Luc-6BS expression plasmid was constructed with a luciferase coding gene (SEQ ID NO: 35) and a ZFP9 binding site (SEQ ID NO: 34). The construction method was the same as in 5.3 above. 4 nmol of TINNeL-ZFP9 or other control proteins TINNe-ZFP9, T-ZFP9 was mixed with 5 μg of pTT5-Luc-6BS expression plasmid for 30 minutes, and then injected into the left leg muscle of nude mice, and the fluorescence intensity was imaged and analyzed 24 hours later. The results are shown in Figure 24. Almost no fluorescence was detected in the T-ZFP9 group and the non-treated group, while weak fluorescence was detected in TINNe-ZFP9, while strong fluorescence was detected in the TINNeL-ZFP9 group. Therefore, it was confirmed that TINNeL-ZFP9 can effectively bind to and deliver DNA in mice.

[0177] Although specific embodiments of the invention are described in detail, those skilled in the art will understand in light of all the teachings published herein that various modifications and changes can be made to the details, and that all such modifications are within the scope of the invention, the full scope of which is given by the appended claims and their equivalents. This specification discloses the following inventions. [1] A fusion polypeptide comprising a multimerization domain sequence, a cell membrane-permeable peptide, a pH-sensitive membrane fusion peptide, and a protease recognition sequence. [2] the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or any higher order multimerization domain; Preferably, the multimerization domain is selected from the group consisting of leucine zipper, NOE, GCN4-P1, delta, and any combination thereof; Preferably, the multimerization domain is selected from leucine zippers, Preferably, the multimerization domain sequence comprises the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The fusion polypeptide according to [1]. [3] The cell membrane penetrating peptide is selected from the group consisting of penetratin, Tat-derived peptides (e.g., Tat(48-60) or Tat(47-57)), Rev(34-50), VP22, transportan, Pep-1, Pep-7, and any combination thereof; Preferably, the cell membrane penetrating peptide comprises a Tat-derived peptide such as Tat(48-60), Preferably, the cell membrane-permeable peptide comprises the sequence shown in SEQ ID NO: 14. The fusion polypeptide according to [1] or [2]. [4] the pH-sensitive fusogenic peptide is selected from the group consisting of influenza virus HA2 or a mutant thereof (e.g., INF7, KALA, or GALA), melittin, and any combination thereof; Preferably, the pH-sensitive membrane fusogenic peptide comprises INF7, Preferably, the pH-sensitive membrane fusion peptide is the fusion polypeptide according to any one of [1] to [3], which comprises the sequence shown in SEQ ID NO: 12. [5] the protease is selected from furin and / or lysosomal cysteine ​​proteases; The fusion polypeptide according to any one of [1] to [4], wherein the protease recognition sequence is selected from the group consisting of a furin recognition sequence, a lysosomal cysteine ​​protease recognition sequence, and a combination thereof. [6] The furin recognition sequence is RX 1 -X 2 -R (SEQ ID NO: 47), wherein X 1 is any amino acid, and X 2 is K or R, Preferably, the furin recognition sequence is RRX 1 -X 2 -R (SEQ ID NO: 48), Preferably, the furin recognition sequence comprises the sequence shown in SEQ ID NO: 49, Preferably, the furin recognition sequence comprises the sequence shown in SEQ ID NO:8. [7] the lysosomal cysteine ​​protease is selected from the group consisting of cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D, or cathepsin H; Preferably, the lysosomal cysteine ​​protease is cathepsin L; Preferably, the cathepsin L recognition sequence comprises the sequence shown in SEQ ID NO: 10. The fusion polypeptide according to [5] or [6]. [8] the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence; Preferably, the protease recognition sequence comprises SEQ ID NO:49 and SEQ ID NO:10, Preferably, the protease recognition sequence includes SEQ ID NO: 8 and SEQ ID NO: 10. The fusion polypeptide according to any one of [1] to [7]. [9] The fusion polypeptide comprises, from the N-terminus to the C-terminus, the cell membrane penetrating peptide, the pH-sensitive membrane fusogenic peptide, and the protease recognition sequence, or comprises, from the N-terminus to the C-terminus, the pH-sensitive membrane fusogenic peptide, the cell membrane penetrating peptide, and 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 described above; The fusion polypeptide according to any one of [1] to [8], wherein the protease recognition sequence comprises, from the N-terminus to the C-terminus, the furin recognition sequence and the cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, the cathepsin L recognition sequence and the furin recognition sequence.

[10] any two adjacent domains contained in said fusion polypeptide are optionally linked by a peptide linker; Preferably, the peptide linker is (G m S) n wherein m is selected from integers of 1 to 4, and n is selected from integers of 1 to 3.

[11] The fusion polypeptide according to any one of [1] to

[10] , comprising any one of the sequences shown in SEQ ID NO:16 to SEQ ID NO:18.

[12] A multimer of the fusion polypeptide according to any one of [1] to

[11] , Preferably, the multimer is a dimer, trimer, or tetramer; Preferably, the multimer is a homomultimer.

[13] A fusion protein comprising the fusion polypeptide according to any one of [1] to

[11] and an additional polypeptide, Preferably, the additional polypeptide comprises a detectable label, Preferably, the additional polypeptide is a fusion protein comprising an epitope tag, a protein sequence encoded by a reporter gene, and / or a nuclear localization signal (NLS) sequence.

[14] the additional polypeptide is a nucleic acid binding domain sequence; Preferably, the nucleic acid binding domain sequence is a zinc finger protein (e.g., ZFP9); Preferably, the nucleic acid binding domain sequence comprises the sequence shown in SEQ ID NO: 31, Preferably, the fusion protein comprises any one of the sequences shown in SEQ ID NO: 19 to SEQ ID NO: 21. The fusion protein according to

[13] .

[15] (i) the fusion protein comprises, from the N-terminus to the C-terminus, a cell membrane penetrating peptide, a pH-sensitive membrane fusogenic peptide, a protease recognition sequence, and the additional polypeptide, and a multimerization domain sequence is located at the N-terminus or C-terminus of the fusion protein or between any two adjacent domains described above, and preferably, the protease recognition sequence comprises, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, the cathepsin L recognition sequence and the furin recognition sequence, or (ii) the fusion protein comprises, from the N-terminus to the C-terminus, the pH-sensitive membrane fusogenic peptide, the cell membrane penetrating peptide, the protease recognition sequence, and the additional 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 described above, and preferably, the protease recognition sequence comprises, from the N-terminus to the C-terminus, the furin recognition sequence and the cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, the cathepsin L recognition sequence and the furin recognition sequence, or (iii) The fusion protein according to

[13] or

[14] , wherein the additional polypeptide is fused to the C-terminus of the fusion polypeptide.

[16] A multimer of the fusion protein according to any one of

[13] to

[15] , Preferably, the multimer is a dimer, trimer, or tetramer; Preferably, the multimer is a homomultimer.

[17] An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion polypeptide according to any one of [1] to

[11] or the fusion protein according to any one of

[13] to

[15] .

[18] A vector comprising the isolated nucleic acid molecule described in

[17] .

[19] A host cell comprising the isolated nucleic acid molecule according to

[17] or the vector according to

[18] .

[20] A method for preparing a fusion polypeptide according to any one of [1] to

[11] or a fusion protein according to any one of

[13] to

[15] , the method comprising culturing a host cell according to

[19] under appropriate conditions and recovering the fusion polypeptide or the fusion protein from the cell culture, wherein the fusion polypeptide or the fusion protein exists as a multimer.

[21] A complex comprising the multimer according to

[12] or the multimer according to

[16] and a cargo molecule, Preferably, the cargo molecule is selected from the group consisting of a protein, a nucleic acid, a carbohydrate, a lipid, a chemical compound, and any mixture thereof; Preferably, said cargo molecule is fused, chemically coupled or non-covalently associated to said multimer complex.

[22] the cargo molecule is a peptide or a protein; Preferably, the cargo molecule is fused to the multimer.

[23] the cargo molecule is a nucleic acid; Preferably, the nucleic acid is selected from the group consisting of a DNA molecule, an RNA molecule, an siRNA, an antisense oligonucleotide, a ribozyme, an aptamer, and any combination thereof; Preferably, the multimer is a multimer of the fusion protein described in

[14] .

[24] the multimer is chemically coupled to the cargo molecule; Preferably, the chemical coupling is achieved by 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.

[25] the multimer is non-covalently linked to the cargo molecule; Preferably, the multimer is electrostatically conjugated to the cargo molecule.

[26] A pharmaceutical composition comprising the complex according to any one of

[21] to

[25] and a pharma- ceutically acceptable carrier and / or excipient, Preferably, the cargo molecule comprises a pharma- ceutical active agent or a detectable label.

[27] Use of the conjugate according to any one of

[21] to

[25] or the pharmaceutical composition according to

[26] in the manufacture of a medicament for treating a disease, wherein a cargo molecule contained in the conjugate is capable of treating the disease; Preferably, the disease is a disease associated with programmed necrosis and the cargo molecule comprises protein phosphatase 1B, and preferably, the disease associated with programmed necrosis comprises liver damage (e.g., drug-induced liver injury), inflammatory diseases, ischemia-reperfusion injury, and / or neurodegenerative diseases.

[28] A method for treating a disease, comprising administering to a subject in need thereof a complex according to any one of

[21] to

[25] , wherein a cargo molecule contained in the complex is capable of treating the disease; Preferably, the disease is a disease associated with programmed necrosis and the cargo molecule comprises protein phosphatase 1B, and preferably, the disease associated with programmed necrosis comprises liver damage (e.g., drug-induced liver injury), inflammatory diseases, ischemia-reperfusion injury, and / or neurodegenerative diseases.

[29] A kit comprising the fusion polypeptide according to any one of [1] to

[11] , the multimer according to

[12] , the fusion protein according to any one of

[13] to

[15] , the multimer according to

[16] , the isolated nucleic acid molecule according to

[17] , the vector according to

[18] , the host cell according to

[19] , or the complex according to any one of

[21] to

[25] , Preferably, the kit further comprises instructions for transfection and / or intracellular delivery.

[30] Use of a fusion polypeptide according to any one of [1] to

[11] , a multimer according to

[12] , a fusion protein according to any one of

[13] to

[15] , a multimer according to

[16] , an isolated nucleic acid molecule according to

[17] , a vector according to

[18] , a host cell according to

[19] , or a complex according to any one of

[21] to

[25] as a delivery reagent.

[31] A method for delivering a cargo molecule into a cell, the method comprising contacting the cell with the complex according to any one of

[21] to

[25] , wherein the cargo molecule is a cargo molecule contained in the complex; Preferably, the contacting of the cell with the complex is carried out in vitro; Preferably, said cargo molecule is selected from the group consisting of a nucleic acid, a peptide or protein, a carbohydrate, a lipid, a chemical compound, and any mixture thereof, preferably, said nucleic acid is selected from the group consisting of a DNA molecule, an RNA molecule, an siRNA, an antisense oligonucleotide, a ribozyme, an aptamer, and any combination thereof.

Claims

1. A fusion polypeptide comprising a multimerization domain sequence, a cell membrane penetrating peptide, a pH-sensitive membrane fusogenic peptide, and a protease recognition sequence, the multimerization domain is selected from the group consisting of leucine zipper, NOE, delta, and any combination thereof, wherein the leucine zipper comprises a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2; The fusion polypeptide comprises, from the N-terminus to the C-terminus, the cell membrane-permeable peptide, the pH-sensitive membrane fusion peptide, and the protease recognition sequence, or comprises, from the N-terminus to the C-terminus, the pH-sensitive membrane fusion peptide, the cell membrane-permeable peptide, and the protease recognition sequence, and the multimerization domain sequence is located at the N-terminus or C-terminus of the fusion polypeptide.

2. The fusion polypeptide described in claim 1, wherein the multimerization domain is selected from a leucine zipper and the multimerization domain sequence includes the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The fusion polypeptide according to claim 1 or 2, wherein the cell membrane permeable 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.

4. A fusion polypeptide described in any one of claims 1 to 3, wherein the cell membrane-permeable peptide comprises a Tat-derived peptide.

5. The fusion polypeptide of claim 4, wherein the Tat-derived peptide is Tat(48-60).

6. A fusion polypeptide described in any one of claims 1 to 5, wherein the cell membrane-permeable peptide includes the sequence shown in sequence number 14.

7. The fusion polypeptide according to any one of claims 1 to 6, wherein the pH-sensitive membrane fusion peptide is selected from the group consisting of influenza virus HA2 or a mutant thereof, melittin, and any combination thereof.

8. A fusion polypeptide described in any one of claims 1 to 7, wherein the pH-sensitive membrane fusion peptide includes INF7.

9. A fusion polypeptide described in any one of claims 1 to 8, wherein the pH-sensitive membrane fusion peptide comprises the sequence shown in SEQ ID NO:

12.

10. The fusion polypeptide according to any one of claims 1 to 9, wherein the protease is selected from furin and / or lysosomal cysteine ​​proteases.

11. A fusion polypeptide described in any one of claims 1 to 10, wherein the protease recognition sequence is selected from the group consisting of a furin recognition sequence, a lysosomal cysteine ​​protease recognition sequence, and a combination thereof.

12. The furin recognition sequence is R-X 1 -X 2 -R (SEQ ID NO: 47), where X 1 is any amino acid, and X 2 is K or R.

13. The fusion polypeptide according to claim 12, wherein the furin recognition sequence comprises R-R-X 1 -X 2 -R (SEQ ID NO:48).

14. The fusion polypeptide described in claim 12, wherein the furin recognition sequence includes the sequence shown in SEQ ID NO:49 or SEQ ID NO:

8.

15. The fusion polypeptide of any one of claims 10 to 14, wherein the lysosomal cysteine ​​protease is selected from the group consisting of cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D, or cathepsin H.

16. A fusion polypeptide described in any one of claims 10 to 15, wherein the lysosomal cysteine ​​protease is cathepsin L.

17. A fusion polypeptide described in claim 15 or 16, wherein the cathepsin L recognition sequence includes the sequence shown in sequence number 10.

18. The fusion polypeptide according to any one of claims 1 to 17, wherein the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence.

19. A fusion polypeptide described in any one of claims 1 to 18, wherein the protease recognition sequence includes SEQ ID NO:49 and SEQ ID NO:10, or SEQ ID NO:8 and SEQ ID NO:

10.

20. A fusion polypeptide described in any one of claims 1 to 19, wherein the protease recognition sequence comprises, from the N-terminus to the C-terminus, the furin recognition sequence and the cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, the cathepsin L recognition sequence and the furin recognition sequence.

21. The fusion polypeptide according to any one of claims 1 to 20, wherein any two adjacent domains contained in the fusion polypeptide are linked by a peptide linker.

22. The fusion polypeptide of claim 21, wherein the peptide linker is (G m S) n , where m is selected from an integer of 1 to 4, and n is selected from an integer of 1 to 3.

23. The fusion polypeptide according to any one of claims 1 to 22, wherein the fusion polypeptide comprises a sequence as shown in any one of SEQ ID NOs: 16 to 18.

24. A multimer of a fusion polypeptide according to any one of claims 1 to 23.

25. The multimer described in claim 24, wherein the multimer is a dimer, trimer, or tetramer.

26. The multimer described in claim 24 or 25, wherein the multimer is a homomultimer.

27. A fusion protein comprising a fusion polypeptide according to any one of claims 1 to 23 and an additional polypeptide.

28. The fusion protein of claim 27, wherein the additional polypeptide comprises a detectable label, and the additional polypeptide comprises an epitope tag, a protein sequence encoded by a reporter gene, and / or a nuclear localization signal (NLS) sequence.

29. The fusion protein of claim 27 , wherein the additional polypeptide is a nucleic acid binding domain sequence.

30. The fusion protein of claim 29, wherein the nucleic acid binding domain sequence is a zinc finger protein that is ZEP9, or the nucleic acid binding domain sequence comprises the sequence shown in SEQ ID NO:

31.

31. The fusion protein described in claim 27, comprising an array shown in any one of SEQ ID NOs: 19 to 21.

32. (i) the fusion protein comprises, from the N-terminus to the C-terminus, a cell membrane penetrating peptide, a pH-sensitive membrane fusogenic peptide, a protease recognition sequence, and the additional polypeptide, and the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion protein, and the protease recognition sequence comprises, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or comprises, from the N-terminus to the C-terminus, the cathepsin L recognition sequence and the furin recognition sequence; or (ii) the fusion protein comprises, from the N-terminus to the C-terminus, the pH-sensitive membrane fusogenic peptide, the cell membrane penetrating peptide, the protease recognition sequence, and the additional polypeptide, the multimerization domain sequence being located at the N-terminus or C-terminus of the fusion protein, the protease recognition sequence comprising, from the N-terminus to the C-terminus, the furin recognition sequence and the cathepsin L recognition sequence, or comprising, from the N-terminus to the C-terminus, the cathepsin L recognition sequence and the furin recognition sequence, and (iii) The fusion protein of any one of claims 27 to 31, wherein the additional polypeptide is fused to the C-terminus of the fusion polypeptide.

33. A multimer of the fusion protein according to any one of claims 27 to 32.

34. The multimer of claim 33, wherein the multimer is a dimer, trimer, or tetramer.

35. The multimer described in claim 33 or 34, wherein the multimer is a homomultimer.

36. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a fusion polypeptide according to any one of claims 1 to 23 or a fusion protein according to any one of claims 27 to 32.

37. 37. A vector comprising the isolated nucleic acid molecule of claim 36.

38. 38. A host cell comprising the isolated nucleic acid molecule of claim 36 or the vector of claim 37.

39. A method for preparing a fusion polypeptide according to any one of claims 1 to 23 or a fusion protein according to any one of claims 27 to 32, comprising culturing a host cell according to claim 38 under suitable conditions and recovering said fusion polypeptide or said fusion protein from the cell culture, wherein said fusion polypeptide or said fusion protein is present as a multimer.

40. A complex comprising the multimer according to any one of claims 24 to 26 or the multimer according to any one of claims 33 to 35, and a cargo molecule.

41. The complex of claim 40, wherein the cargo molecule is selected from the group consisting of proteins, nucleic acids, carbohydrates, lipids, chemical compounds, and any mixture thereof.

42. A complex described in claim 40 or 41, wherein the cargo molecule is fused to, chemically coupled to, or non-covalently linked to the multimer.

43. The complex of any one of claims 40 to 42, wherein the cargo molecule is a peptide or a protein, and the cargo molecule is fused to the multimer.

44. The complex of any one of claims 40 to 42, wherein the cargo molecule is a nucleic acid, the nucleic acid being selected from the group consisting of a DNA molecule, an RNA molecule, an siRNA, an antisense oligonucleotide, a ribozyme, an aptamer, and any combination thereof, and the multimer is a multimer of a fusion protein of any one of claims 29 to 31.

45. 43. The conjugate of any one of claims 40 to 42, wherein the multimer is chemically coupled to the cargo molecule, the chemical coupling being achieved by 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.

46. The complex of any one of claims 40 to 42, wherein the multimer is electrostatically conjugated to the cargo molecule.

47. A pharmaceutical composition comprising the conjugate according to any one of claims 40 to 46 and a pharma- ceutically acceptable carrier and / or excipient.

48. The pharmaceutical composition described in claim 47, wherein the cargo molecule comprises a pharma- ceutically active substance or a detectable label.

49. 50. Use of a conjugate according to any one of claims 40 to 46 or a pharmaceutical composition according to claim 47 or 48 in the manufacture of a medicament for the treatment of a disease, wherein a cargo molecule comprised in said conjugate is capable of treating said disease.

50. The use described in claim 49, wherein the disease is a disease associated with programmed necrosis and the cargo molecule comprises protein phosphatase 1B.

51. The use described in claim 50, wherein the disease associated with programmed necrosis includes liver damage, inflammatory diseases, ischemia-reperfusion injury, and / or neurodegenerative diseases.

52. The use described in claim 51, wherein the liver damage is drug-induced liver damage.

53. A complex according to any one of claims 40 to 46 for use in the treatment of a disease, wherein a cargo molecule contained in the complex is capable of treating the disease.

54. The complex of claim 53, wherein the disease is a disease associated with programmed necrosis and the cargo molecule comprises protein phosphatase 1B.

55. The complex of claim 54, wherein the disease associated with programmed necrosis includes liver damage, inflammatory diseases, ischemia-reperfusion injury, and / or neurodegenerative diseases.

56. The complex described in claim 55, wherein the liver damage is drug-induced liver damage.

57. A kit comprising a fusion polypeptide according to any one of claims 1 to 23, a multimer according to any one of claims 24 to 26, a fusion protein according to any one of claims 27 to 32, a multimer according to any one of claims 33 to 35, an isolated nucleic acid molecule according to claim 36, a vector according to claim 37, a host cell according to claim 38, or a complex according to any one of claims 40 to 46, The kit further comprises instructions for transfection and / or intracellular delivery.

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