MULTIMERIZATION DELIVERY SYSTEM FOR INTRACELLULAR DELIVERY OF MOLECULE

Abstract

A multimerization delivery system that can be used to deliver a cargo molecule intracellularly. The multimerization delivery system can achieve high-efficiency endocytosis of a cargo molecule and high-efficiency release thereof from an endocytic vesicle, significantly improving the cytoplasmic delivery efficiency of the cargo molecule. Once the cargo molecule is available in the cytoplasm, the cargo molecule can play any role related thereto. The multimerization delivery system provides an effective means for affecting the biological mechanisms and pathways of cells, and can be used in various fields such as research, treatment, and diagnosis.

Claims

1. A fusion polypeptide, which comprises a multimerization domain sequence, a cell-penetrating peptide, a pH-sensitive fusogenic peptide, and a protease recognition sequence.

2. The fusion polypeptide according to claim 1, wherein 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 zipper; preferably, the multimerization domain sequence comprises the sequence shown in SEQ ID NO: 1 or 2.

3. The fusion polypeptide according to claim 1 or 2, wherein the cell-penetrating peptide is selected from the group consisting of Penetratin, a Tat-derived peptide (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-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60); preferably, the cell-penetrating peptide comprises the sequence shown in SEQ ID NO: 14.

4. The fusion polypeptide according to any one of claims 1 to 3, wherein the pH-sensitive fusogenic peptide is selected from the group consisting of influenza virus HA2 or mutant thereof (e.g., INF7, KALA or GALA), melittin, and any combination thereof; preferably, the pH-sensitive fusogenic peptide comprises INF7; preferably, the pH-sensitive fusogenic peptide comprises the sequence shown in SEQ ID NO:12.

5. The fusion polypeptide according to any one of claims 1 to 4, wherein the protease is selected from furin and/or lysosomal cysteine protease; preferably, the protease recognition sequence is selected from the group consisting of furin recognition sequence, lysosomal cysteine protease recognition sequence, and combination thereof.

6. The fusion polypeptide according to claim 5, wherein the furin recognition sequence comprises R-X.sub.1-X.sub.2-R (SEQ ID NO: 47), wherein X.sub.1 is any amino acid, and X.sub.2 is K or R; preferably, the furin recognition sequence comprises R-R-X.sub.1-X.sub.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 fusion polypeptide according to claim 5 or 6, 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; preferably, the lysosomal cysteine protease is cathepsin L; preferably, the cathepsin L recognition sequence comprises the sequence shown in SEQ ID NO: 10.

8. The fusion polypeptide according to any one of claims 1 to 7, wherein 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 comprises SEQ ID NO: 8 and SEQ ID NO: 10.

9. The fusion polypeptide according to any one of claims 1 to 8, wherein the fusion polypeptide comprises: the cell-penetrating peptide, the pH-sensitive fusogenic peptide, the protease recognition sequence from the N-terminus to the C-terminus, or, comprises: the pH-sensitive fusogenic peptide, the cell-penetrating peptide, the protease recognition sequence from the N-terminus to the C-terminus; and the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion polypeptide, or between any two adjacent domains as above mentioned; preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

10. The fusion polypeptide according to any one of claims 1 to 9, wherein any two adjacent domains contained in the fusion polypeptide are optionally linked by a peptide linker; preferably, the peptide linker is (G.sub.mS).sub.n, wherein m is selected from an integer of 1 to 4 and n is selected from an integer of 1 to 3.

11. The fusion polypeptide according to any one of claims 1 to 10, wherein the fusion polypeptide comprises the sequence shown in any one of SEQ ID NOs: 16 to 18.

12. A multimer of the fusion polypeptide according to any one of claims 1 to 11; preferably, the multimer is a dimer, trimer or tetramer; preferably, the multimer is a homomultimer.

13. A fusion protein, which comprises the fusion polypeptide according to any one of claims 1 to 11, and an additional polypeptide; preferably, the additional polypeptide comprises a detectable label; preferably, the additional polypeptide comprises an epitope tag, a protein sequence encoded by reporter gene and/or a nuclear localization signal (NLS) sequence.

14. The fusion protein according to claim 13, wherein 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 the sequence shown in any one of SEQ ID NOs: 19 to 21.

15. The fusion protein according to claim 13 or 14, wherein, (i) the fusion protein comprises: the cell-penetrating peptide, the pH-sensitive fusogenic peptide, the protease recognition sequence and the additional polypeptide from the N-terminus to the C-terminus; and, the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion protein, or between any two adjacent domains described above; preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus; or, (ii) the fusion protein comprises: the pH-sensitive fusogenic peptide, the cell-penetrating peptide, the protease recognition sequence, and the additional polypeptide from the N-terminus to the C-terminus; and, the multimerization domain sequence is located at the N-terminus or the C-terminus of the fusion protein, or between any two adjacent domains described above; preferably, the protease recognition sequence comprises the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or comprises the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus; or, (iii) 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 claims 13 to 15; preferably, the multimer is a dimer, trimer or tetramer; preferably, the multimer is a homomultimer.

17. An isolated nucleic acid molecule, which comprises a nucleotide sequence encoding the fusion polypeptide according to any one of claims 1 to 11, or the fusion protein according to any one of claims 13 to 15.

18. A vector, which comprises the isolated nucleic acid molecule according to claim 17.

19. A host cell, which comprises the isolated nucleic acid molecule according to claim 17 or the vector according to claim 18.

20. A method for preparing the fusion polypeptide according to any one of claims 1 to 11, or the fusion protein according to any one of claims 13 to 15, which comprises, culturing the host cell according to claim 19 under suitable conditions, and recovering the fusion polypeptide or the fusion protein from a cell culture, wherein the fusion polypeptide or the fusion protein exists as a multimer.

21. A complex, which comprises the multimer according to claim 12 or the multimer according to claim 16, and a cargo molecule; preferably, the cargo molecule is selected from the group consisting of protein, nucleic acid, carbohydrate, lipid, chemical compound and any mixture thereof; preferably, the cargo molecule is fused, chemically coupled or non-covalently linked to the multimer.

22. The complex according to claim 21, wherein the cargo molecule is a peptide or a protein; preferably, the cargo molecule is fused to the multimer.

23. The complex according to claim 21, wherein the cargo molecule is a nucleic acid; preferably, the nucleic acid is selected from the group consisting of DNA molecule, RNA molecule, siRNA, antisense oligonucleotide, ribozyme, aptamer and any combination thereof; preferably, the multimer is a multimer of the fusion protein according to claim 14.

24. The complex according to claim 21, wherein the multimer is chemically coupled to the cargo molecule; preferably, the chemical coupling is achieved through 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 complex according to claim 21, wherein the multimer is non-covalently linked to the cargo molecule; preferably, the multimer is electrostatically conjugated to the cargo molecule.

26. A pharmaceutical composition, which comprises the complex according to any one of claims 21 to 25, and a pharmaceutically acceptable carrier and/or excipient; preferably, the cargo molecule is a pharmaceutically active agent or a detectable label.

27. Use of the complex according to any one of claims 21 to 25 or the pharmaceutical composition according to claim 26 in the manufacture of a medicament for the treatment of a disease; wherein the 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; preferably, the disease associated with programmed necrosis comprises liver injury (e.g., drug-induced liver injury), inflammatory disease, ischemia-reperfusion injury and/or neurodegenerative disease.

28. A method for treating a disease, comprising administering the complex according to any one of claims 21 to 25 to a subject in need thereof; wherein, the 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; preferably, the disease associated with programmed necrosis comprises liver injury (e.g., drug-induced liver injury), inflammatory disease, ischemia-reperfusion injury and/or neurodegenerative disease.

29. A kit, which comprises the fusion polypeptide according to any one of claims 1 to 11, the multimer according to claim 12, the fusion protein according to any one of claims 13 to 15, the multimer according to claim 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the host cell according to claim 19, or the complex according to any one of claims 21 to 25; preferably, the kit further comprises an instruction for transfection and/or intracellular delivery.

30. Use of the fusion polypeptide according to any one of claims 1 to 11, the multimer according to claim 12, the fusion protein according to any one of claims 13 to 15, the multimer according to claim 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the host cell according to claim 19, or the complex according to any one of claims 21 to 25 as a delivery reagent.

31. A method for delivering a cargo molecule into a cell, comprising: contacting the cell with the complex according to any one of claims 21 to 25, wherein the cargo molecule is the cargo molecule contained in the complex; preferably, the contacting of the cell with the complex is performed in vitro; preferably, the cargo molecule is selected from the group consisting of nucleic acid, peptide or protein, carbohydrate, lipid, chemical compound and any mixture thereof preferably, the nucleic acid is selected from the group consisting of DNA molecule, RNA molecule, siRNA, antisense oligonucleotide, ribozyme, aptamer, and any combination thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0137] FIG. 1 shows the clone design for the multimerization delivery system-eGFP based on leucine zipper.

[0138] FIG. 2 shows the effect of leucine zipper on the state of protein aggregate.

[0139] FIG. 3 shows the evaluation of the endocytosis efficiency and vesicle escape efficiency of the delivery system after the addition of leucine zipper domain.

[0140] FIG. 4 shows the evaluation of the endocytosis efficiency of the delivery system under serum conditions after the addition of leucine zipper domain.

[0141] FIG. 5 shows the clone design for the delivery system-eGFP based on multiple multimerization domains.

[0142] FIG. 6 shows the fluorescence microscopy imaging of the effects of various multimerization domains on endocytosis efficiency.

[0143] FIG. 7 shows the evaluation of the effects of various multimerization domains on endocytic efficiency.

[0144] FIG. 8 shows the clone design for TINNeL-eGFP/GFPβ1-10.

[0145] FIG. 9 shows the evaluation of the endocytosis efficiency of TINNeL delivery system.

[0146] FIG. 10 shows the evaluation of the vesicle escape efficiency of TINNeL delivery system.

[0147] FIG. 11 shows the evaluation of the delivery efficiency of TINNeL delivery system under different serum concentrations.

[0148] FIG. 12 shows a schematic diagram of the transport mechanism of the multimerization delivery system of the present invention.

[0149] FIG. 13 shows the clone constructions for the protein of TINNeL-Ppm1b delivery system and other control proteins.

[0150] FIG. 14 shows the effect of TINNeL-Ppm1b on inhibiting TNF-α-induced cell necrosis.

[0151] FIG. 15 shows the survival rate of mice after inhibition of TNF-α-induced death by TINNeL-Ppm1b.

[0152] FIG. 16 shows the HE staining of mouse SIRS cecum caused by TNF-α after inhibition by TINNeL-Ppm1b.

[0153] FIG. 17 shows the injury scores of mouse SIRS cecum caused by TNF-α after inhibition by TINNeL-Ppm1b.

[0154] FIG. 18 shows the changes in ALT/AST levels after TINNeL-Ppm1b treatment of APAP-induced acute liver injury in mice.

[0155] FIG. 19 shows the survival rate of mice with TINNeL-Ppm1b treatment against death caused by APAP.

[0156] FIG. 20 shows the clone constructions for the protein of TINNeL-ZFP9 delivery system and other control proteins.

[0157] FIG. 21 shows a schematic diagram of the eukaryotic expression plasmid structure for ZFP9 delivery.

[0158] FIG. 22 shows the flow cytometry analysis about the transduction efficiency of red fluorescent protein expression plasmid by TINNeL-ZFP9.

[0159] FIG. 23 shows the flow cytometry analysis about the red fluorescent protein expression plasmid transduced by TINNeL-ZFP9 and X-tremeGNEN under serum conditions.

[0160] FIG. 24 shows the image of mice about fluorescence expression of Luciferase plasmid transduced by TINNeL-ZFP9.

[0161] FIG. 25 shows fluorescence microscopy observations of transduction of GFPβ1-10 by the delivery system.

[0162] SEQUENCE INFORMATION

[0163] Information of the partial sequences involved in the present invention is provided in Table 1 below.

TABLE-US-00001 TABLE 1 Description of sequences SEQ ID NO Description 1 Leucine zipper-1 2 Leucine zipper-2 3 NOE multimerization domain 4 GCN4-P1 multimerization domain 5 Delta multimerization domain 6 Nucleic acid sequence encoding leucine zipper-1 7 Nucleic acid sequence encoding leucine zipper-2 8 Furin recognition sequence Ne 9 Nucleic acid sequence encoding Ne 10 CTSL recognition sequence N 11 Nucleic acid sequence encoding N 12 INF7 13 Nucleic acid sequence encoding INF7 14 Tat(48-60) 15 Nucleic acid sequence encoding Tat(48-60) 16 Fusion protein TINL 17 Fusion protein TINeL 18 Fusion protein TINNeL 19 Fusion protein TINL-ZFP9 20 Fusion protein TINeL-ZFP9 21 Fusion protein TINNeL-ZFP9 22 NLS 23 eGFP 24 Nucleic acid sequence encoding eGFP 25 GFPβ1-10-NLS 26 Nucleic acid sequence encoding GFPβ1-10-NLS 27 Nucleic acid sequence encoding Histone-H3 28 Nucleic acid sequence encoding GFPβ11 29 Ppm1b 30 Nucleic acid sequence encoding Ppm1b 31 ZFP9-NLS 32 Nucleic acid sequence encoding ZFP9-NLS 33 tdTomato coding sequence 34 ZFP9 binding site 6* 35 Luciferase coding gene 36 Influenza virus HA2 37 KALA 38 GALA 39 Melittin 40 Penetratin 41 HIV-TAT(47-57) 42 HIV-1 Rev(34-50) 43 VP22 44 Transportan 45 Pep-1 46 Pep-7 47 Furin recognition sequence-1 48 Furin recognition sequence-2 49 Furin recognition sequence-3 50 Peptide linker

EXAMPLES

[0164] The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit, the present invention.

[0165] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods in the present invention were performed basically 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 F. M. Ausubel et al., Refined Molecular Biology Laboratory Manual, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes were used according to the conditions recommended by the product manufacturer. Those skilled in the art appreciate that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention as claimed.

[0166] The sources of the main reagents involved in the following examples are as follows:

[0167] The materials required for clone construction related reagents 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);

[0168] 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);

[0169] The media required for protein purification were as follows: SP SEPHAROSE FAST FLOW (GE Healthcare, 17-0729-01), NI SEPHAROSE (GE Healthcare, 17-5268-02);

[0170] The reagents required for protein purification and preservation were as follows: glycerol/glycerol/C.sub.3H.sub.8O.sub.3 (SIGMA-ALDRICH, G5516), KCl (Xilong Chemical Industry, 1002007), Na.sub.2HPO.sub.4.12H.sub.2O (Xilong Chemical Industry, 1001067AR), KH.sub.2PO.sub.4 (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);

[0171] Reagents required for cell culture: FBS (GIBCO, 10099-133), DMEM (GIBCO, 11965092), trypsin (AMRESCO, 0458);

[0172] 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-b1-5b), polybrene (Santa Cruz, sc-134220);

[0173] The GFPβ1-10, ZFP9, Ppm1b, dsRed, mCherry, Histone-H3 related plasmids used in the experiment were synthesized by Sangon Bio, and the plasmid used for amplifying the Cas9 sequence was pCasKP-hph (Addgene, 117232);

[0174] Other reagents: TNF-α (Novoprotein, CF09), PI (ThmeroFisher, P3566)

[0175] Cell lines: HEK-293T (human kidney epithelial cells), L929 (mouse fibroblasts) were purchased from ATCC.

Example 1: Establishment and Performance Evaluation of TL Multimerization Delivery System

[0176] In this example, a multimerization delivery system was established by using leucine zipper. The effect of the multimerization on endocytosis efficiency was observed by delivering green fluorescent protein eGFP. The effect of the multimerization on vesicle escape efficiency was observed by delivering GFP β1-10-NLS (briefly referred as GFP β-10) based on the Split-GFP evaluation method. Thereby, comprehensive evaluation of the effects of the multimerization on the delivery efficiency of the delivery system was carried out. At the same time, by adding serum (FBS) to the system, the difference in endocytosis efficiency before and after multimerization was observed to evaluate the change of serum tolerance.

[0177] 1.1 Construction of Expression Vector for Multimerization Delivery System (TAT-Leu Zipper)-Cargo Molecule Complex

[0178] An expression vector of recombinant protein containing TAT, Leu-Zipper (leucine zipper), and cargo molecule was constructed, and the structures of the nucleic acids encoding each recombinant protein were shown in FIG. 1. The amino acid sequence of each component from the N-terminus to the C-terminus was shown in the table below. The construction method was as follows: first, the nucleic acid sequences encoding TAT, leucine zipper, and cargo molecule (eGFP or GFP β1-10) in the delivery system were 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 and the overlap on upstream of the corresponding NdeI restriction site in pET21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site in pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00002 TABLE 2 Components contained in the delivery system-cargo molecule complex N-terminal .fwdarw. C-terminal Complex components and their sequences name CPP Leucine zipper Cargo T-eGFP Tat None eGFP TL-eGFP SEQ ID SEQ ID SEQ ID NO: 14 NO: 1 NO: 23 T-GFP None GFP β1- β1-10 10-NLS TL-GFP SEQ ID SEQ ID β1-10 NO: 1 NO: 25

[0179] 1.2 Expression and Purification of Multimerization Delivery System-Cargo Molecule Complex:

[0180] The expression plasmid described in 1.1 was transformed into the expression strain E. coli BL21 (DE3); a single colony was picked from the transformed plate and inoculated into 5 ml of LB liquid medium containing ampicillin resistance and cultivated overnight, and then 1 ml of bacterial liquid overnight cultured was taken, and transferred to 500 ml of LB liquid medium containing ampicillin resistance, and cultivated at 37° C. and 180 rpm until the bacterial liquid had OD.sub.600 of about 0.6, and then the inducer IPTG was added to a final concentration of 0.2 mM, and induced at 25° C. for 8 hours; after the end of the induced expression, the cells were collected after centrifugation under 7000 g at 4° C. for 10 minutes; then the cells were resuspended with 10 ml of protein purification equilibration buffer (PBS, 5% glycerol), and disrupted by ultrasonication. Then, the supernatant was taken by centrifugation and loaded onto the SP strong cation chromatography column of the protein purification system; then the protein purification system was used to elute the target protein with protein elution buffer (PBS+0.2M NaCl˜PBS+0.6M NaCl to remove impurity proteins, PBS+1.6M NaCl to collect the elution product). The protein concentration can be determined according to spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at −20° C.

[0181] 1.3 Evaluation of Multimerization Effect of Leucine Zipper on Delivery System-Cargo Complex

[0182] TAT-eGFP (T-eGFP) obtained in 1.2 above and leucine zipper-bearing TAT-Leu-eGFP (TL-eGFP) were subjected to non-reducing polyacrylamide gel electrophoresis to analyze its aggregate formation state. The results were shown in FIG. 2. The size of T-eGFP protein in monomer state was about 35 kD, while the protein size of TL-eGFP after the addition of leucine zipper was at 70 kD. This experiment showed that the leucine zipper could promote the delivery system protein to form dimer.

[0183] 1.4 Evaluation of Endocytosis Efficiency of Multimerization Delivery System-eGFP Complex

[0184] HEK-293T cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6; the cells were rinsed three times with serum-free DMEM medium, and then TL-eGFP and T-eGFP at 5 μM respectively were added to serum-free medium; after 3 h incubation, rinsing was performed three times with pre-cooled serum-free DMEM containing 20 U/mL heparin to remove the proteins that were adsorbed on the cell membrane and failed to enter the cells, and the cells were collected to perform flow cytometry analysis. The results were shown in FIG. 3. The intracellular fluorescence intensity of the dimerized protein TL-eGFP was 4-5 times that of the monomer (T-eGFP), which proved that the multimerization process improved the endocytosis efficiency of the delivery system.

[0185] 1.5 Evaluation of Vesicle Escape Efficiency of Multimerization Delivery System-GFP β1-10 Complex

[0186] In the Split-GFP system, the 11 β-sheets of GFP were split into a large fragment (β1-10) and a small fragment (β11), both of which lost their fluorescent activity but could spontaneously associate and restore GFP fluorescence property if they met. Based on this, HEK293T cells that could stably express Histone-β11 was constructed, and the GFPβ1-10 with nuclear entry signal (NLS) was used as Cargo for the fusion expression with the multimerization delivery system to be evaluated, and then subjected to transduction of the stable cell line. When GFPβ1-10 was transduced by the delivery system, it could bind to GFPβ11 and generate complete GFP only after successfully escaping from the endocytic vesicle and entering the cytoplasm or nucleus, so that the endocytic vesicle escape efficiency could be evaluated by the proportion and relative fluorescence intensity of ratio of GFP.

[0187] 1.5.1 Construction of HEK293T-GFPβ11 Cell Line

[0188] (1) Construction of Lentiviral Plasmid of GFPβ11 Cell Line:

[0189] The coding sequence of Histone-H3 (SEQ ID NO: 27) was amplified by PCR, and the coding sequence of GFPβ11 (SEQ ID NO: 28) was short and directly designed in the upstream primer. The components were ligated through multiple rounds of PCR, and in the last round of PCR, the Hind III restriction site and the overlap on upstream of the corresponding Hind III restriction site on Lenti vector were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on Lenti vector were introduced at the 3′ end of the fragment through the reverse primer. The Lenti plasmid was subjected to double digestion with Hind III and BamHI. The insert fragments with overlaps were ligated to the digested Lenti vector by GIBSON assembly.

[0190] (2) Lentiviral Packaging, Infection and Resistance Screening of Cell Lines:

[0191] HEK-293T cells were inoculated into a 6-well plate and cultured overnight. Before plasmid transfection, it was ensured that the number of cells per well was about 2*10.sup.7/m1; before transfection, the cells were transferred into serum-free DMEM medium; 1.5 μg of Lenti recombinant plasmid, 0.75 μg of pMDL plasmid, 0.45 μg of pVSV-G plasmid, 0.3 μg of pREV (mass ratio of 5:3:2:1) were added to 300 μl of serum-free DMEM, followed by slowly blowing. 9 μl (1:3) of X-tremeGENE transfection reagent was added and slowly blown, allowed to stand at room temperature for 15 min, and added dropwise into the cell supernatant; after 8 h, culturing was continued by changing the medium to DMEM containing 10% FBS; after 60 h, the cell culture supernatant was collected for later infection.

[0192] HEK-293T cells were inoculated into a 12-well plate and cultured overnight. Before lentivirus infection, it was ensured that the number of cells in each well was about 2*10.sup.6/ml (50% density); the original cell culture supernatant was discarded, 300 μl of lentivirus (moi=3) and 700 μl of 10% FBS DMEM were added, polybrene was added at a concentration of 10 μg/ml, and the cell plate was centrifuged at 2500 rpm for 30 min under aseptic conditions, and then continued to culture.

[0193] After lentivirus infection for 48 hours, the cells were passaged at a ratio of 1/3, and puromycin was added at a concentration of 2.5 μg/ml for resistance screening; the positive cells obtained by screening were cloned to obtain HEK-293T-Hitone-GFPβ11 monoclonal cell line.

[0194] 1.5.2 Detection of Vesicle Escape Efficiency by Split-GFP System

[0195] The above HEK-293T Histone H3-β11 cells (HEK-293T β11 for short) were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6; serum-free DMEM medium was used to rinse the cells three times, and then TL-GFP β1-10 and T-GFP β1-10 at 5 μM respectively were added to the serum-free medium; after incubation for 3 h, a pre-cooled serum-free DMEM containing 20 U/ml heparin was used to perform rinsing three times to remove the proteins that were adsorbed on the cell membrane and failed to enter the cells, and then the culturing was continued for another 9 h after changing the medium to DMEM medium containing serum. And the cells were collected to perform flow cytometry analysis. The results showed that the vesicle escape efficiency of the dimerized protein TL-GFP β1-10 did not change in comparison with the monomeric T-GFP β1-10 (FIG. 3).

[0196] 1.6 Evaluation of Tolerance of Multimerization Delivery System to Serum Conditions

[0197] HEK-293T cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6; the cells were rinsed three times with serum-free DMEM medium, then TL-eGFP and T-eGFP at 5 μM respectively were added to serum-free, 5%, 10%, 20%, 50%, 100% FBS DMEM media; after incubation for 3 h, pre-cooled serum-free DMEM containing 20 U/mL heparin was used for rinsing to remove the proteins that were adsorbed on the cell membrane and failed to enter the cells, then the medium was replaced with serum-free DMEM medium, and the cells were collected for flow cytometry analysis. The results were shown in FIG. 4. With the increase of serum content, the transduction efficiencies of TL-eGFP and T-eGFP decreased, but the transduction efficiency of TL-eGFP was relatively less affected by serum, and at the condition of 50% FBS, there was almost no detectable intracellular green fluorescence signal after the cells ere transduced by T-eGFP, while the dimerized protein TL-eGFP still had a transduction efficiency of 70% compared to that without serum. It could be seen that the multimerization not only improved the endocytosis efficiency of the protein, but also conferred a higher serum tolerance to the protein.

[0198] 1.7 Effect of Different Multimerization Domains on Endocytosis Efficiency of Delivery System

[0199] In this example, the effects of different multimerization domain sequences on the endocytosis efficiency of the delivery system were compared. The components of the delivery system-cargo molecule complexes comprising different multimerization domains were shown in the table below. The expression vector for expressing the above-mentioned complexes was prepared by the methods described in the above 1.1 to 1.2, and the above-mentioned complexes was expressed and purified. The schematic diagram of the constructed plasmid was shown in FIG. 5.

TABLE-US-00003 TABLE 3 Delivery systems comprising different multimerization domains N-terminal .fwdarw. C-terminal components and sequences thereof Name of Multimerization complex CPP domain Cargo T-eGFP Tat None eGFP TL-eGFP SEQ ID NO: 14 Leucine zipper SEQ ID NO: 23 SEQ ID NO: 1 TL2-eGFP Leucine zipper 2 SEQ ID NO: 2 TN-eGFP NOE SEQ ID NO: 3 TG-eGFP GCN4-P1 SEQ ID NO: 4 TD-eGFP Delta SEQ ID NO: 5

[0200] MA104 cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6; 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 at 5 μM respectively were added to serum-free medium; after incubation for 3 h, the cells were rinsed three times with pre-cooled serum-free DMEM containing 20 U/mL heparin to remove the proteins that were adsorbed on the cell membrane but failed to enter the cells, after the medium was replaced with serum-free medium, fluorescence imaging was performed, and the cells were collected for flow cytometry analysis. The fluorescence imaging results were shown in FIG. 6. The intracellular green fluorescence intensity of the T-eGFP group was very low, while the intracellular fluorescence intensity of the other experimental groups added with the multimerization domain was significantly increased. The results of flow cytometry analysis were shown in FIG. 7. Different multimerization domains all could enhance the average fluorescence intensity of eGFP mediated by TAT, that was, improved the efficiency of TAT-mediated endocytosis, and the leucine zipper-based TL-eGFP and TL2-eGFP had the most obvious effect.

Example 2: Establishment and Performance Evaluation of TINNeL Multimerization Delivery System

[0201] In this example, based on the multimerization delivery system of Example 1, pH-sensitive peptide (INF7) and endocytic vesicle protease-specific cleavage site (CTSL protease: N, Furin protease: Ne) were further added to construct TINNEL delivery system, and its endocytic efficiency and vesicle escape efficiency were evaluated.

[0202] 2.1 Construction of Expression Vector for TINNEL-Cargo Molecule Complex

[0203] The expression vector of TINNEL-cargo molecule recombinant protein was constructed, the amino acid sequence of each component contained in each recombinant protein from the N-terminus to the C-terminus was 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 GFP β1-10) in the delivery system were obtained by PCR amplification, and these parts were ligated through multiple rounds of PCR, and in the last round of PCR, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site on pET-21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly. The schematic diagram of the plasmid was shown in FIG. 8.

TABLE-US-00004 TABLE 4 Components of delivery system-cargo molecule complex N-terminal .fwdarw. C-terminal components and sequences thereof Protease pH- recog- Multimer- Name of sensitive nition ization Cargo complex CPP peptide sequence domain molecule TINNe-eGFP TAT INF7 N + Ne None eGFP TINNeL-eGFP SEQ ID SEQ ID Leucine SEQ ID NO: 14 NO: 12 zipper NO: 23 SEQ ID NO: 1 TINNe-GFP None GFP β1- β1-10 10-NLS TINNeL-GFP Leucine SEQ ID β1-10 zipper NO: 25 SEQ ID NO: 1

[0204] 2.2 Expression and Purification of TINNeL-Cargo Complex

[0205] 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 and inoculated into 5 ml of LB liquid medium containing ampicillin resistance for overnight culture, and then 1 ml of the bacterial solution overnight cultured was transferred to 500 mL of LB liquid medium containing ampicillin resistance, and cultivated at 37° C. and 180 rpm until the bacterial liquid had an OD.sub.600 of about 0.6, and then the inducer IPTG was added to reach a final concentration of 0.2 mM, and the induction was performed at 25° C. for 8 hours; after the end of induced expression, the cells were collected by centrifugation under 7000 g at 4° C. for 10 minutes; then the cells were resuspended with 10 ml of protein purification equilibration buffer (PBS, 5% glycerol), and disrupted by ultrasonication. Then, the supernatant was collected by centrifugation and loaded onto the SP strong cation chromatography column of the protein purification system; then the protein purification system was used to elute the target protein with protein elution buffer (PBS+0.2M NaCl˜PBS+0.7M NaCl to remove impurity proteins, PBS+1.8M NaCl to collect the elution product). The protein concentration could be determined according to spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at −20° C.

[0206] 2.3 Evaluation of Intracellular Delivery Efficiency of TINNeL Delivery System

[0207] Evaluation of endocytosis efficiency: HEK-293T was inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells per well was about 2*10.sup.6; 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 respectively at 5 μM were added to serum-free medium; after incubation for 3 h, the pre-cooled serum-free DMEM containing 20 U/mL heparin was used to rinse for three times to remove the proteins that were adsorbed on the cell membrane but failed to enter the cells, and then the cells were collected for flow cytometry analysis. We found that the fluorescence intensity of TINNeL-eGFP after dimerization was 4 times higher, that was, the endocytosis efficiency was significantly improved after the addition of the leucine zipper domain (FIG. 9).

[0208] Evaluation of vesicle escape efficiency: We used the Split-GFP system to further evaluate the delivery efficiency of the TINNeL system by observing changes in its escape efficiency. HEK-293T β11 were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6; the cells were rinsed three times with serum-free DMEM medium, and then the delivery system protein TINNeL-GFP β1-10 and the control protein TINNe-GFP β1-10 respectively at 1 μM were added to serum-free medium; after incubation for 3 h, the pre-cooled serum-free DMEM containing 20 U/mL heparin was used to rinse for three times to remove the proteins that were adsorbed on the cell membrane but failed to enter the cells. The culturing was continued for another 9 h after changing the medium to serum-containing medium (10% FBS), and the cells were collected for flow cytometry analysis. The results were shown in FIG. 10, in which TINNeL-GFP β1-10 had higher fluorescence intensity, indicating that the TINNeL delivery system could improve both endocytosis efficiency and vesicle escape efficiency, and was a more efficient delivery method.

[0209] 2.4 Evaluation of Delivery Efficiency of TINNeL Delivery System Under Serum Conditions

[0210] HEK-293T cells were inoculated into a 12-well cell culture plate and cultured overnight, before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6; the cells were rinsed three times with serum-free DMEM medium, and TINNeL-GFP β1-10 and the control protein TINNe-GFP β1-10 respectively at 5 μM were added to serum-free, 10%, 20%, 50%, and 100% FBS DMEM media; after incubation for 3 h, the pre-cooled serum-free DMEM containing 20 U/mL heparin was used to rinse for four times to remove the proteins that were adsorbed on the cell membrane but failed to enter the cells. The culturing was continued for another 9 h after changing the medium to serum-free DMEM medium, then the cells were collected for flow cytometry analysis. The results were shown in FIG. 11. With the increase of serum content, the fluorescence intensity of TINNeL-GFP β1-10 and TINNe-GFP β1-10 groups decreased, but TINNeL-GFP β1-10 was less affected. Under the condition of 100% FBS concentration, the green fluorescence signal of TINNe-GFP β1-10 group could hardly be detected, while TINNeL-GFP β1-10 still had 70% efficiency compared with that without serum. TINNeL not only had high efficiency delivery ability, and its delivery efficiency would not be greatly affected in the presence of serum. FIG. 12 shows a schematic diagram of the principle of the TINNeL delivery system.

[0211] In addition, the present inventors also evaluated the vesicle escape efficiency of the delivery system containing protease recognition sequence N (TIN-GFP β1-10) and the delivery system containing protease recognition sequence Ne (TINe-GFP β1-10) based on the Split-GFP system. The delivery systems described above differed from TINNe-GFP β1-10 only in that the protease recognition sequence was replaced by N or Ne alone. The results were shown in FIG. 25. Compared with TI-GFP β1-10 or TIN-GFP β1-10 without protease recognition sequence, either the delivery system (TIN-GFP β1-10) that contained the protease recognition sequence N alone, or the delivery system (TINe-GFP β1-10) that contained the protease recognition sequence Ne, the escape efficiency of endocytic vesicles was significantly improved. The above results showed that either the protease recognition sequence N or Ne alone had the ability to improve the vesicle escape efficiency.

Example 3: Application of TINNeL-Ppm1b in Inhibiting Apoptosis Induced by TNF-α

[0212] Cell death can be divided into apoptosis and necrosis. Among them, cell necrosis may lead to cell membrane rupture, swelling and leakage of contents, resulting in a severe inflammatory response, which is mainly controlled by receptor-interaction kinase 3 (RIP3). Under the stimulation of TNF-α, necrosomes containing Rip1 and Rip3 are formed in cells, and Rip3 in the necrosomes recruits and phosphorylates Mlk1. Phosphorylated Mlk1 translocates to the cell membrane to perform necroptosis, during which the phosphorylation of Rip3 is necessary for the recruitment of Mlk1 to necroptosis. Therefore, the phosphorylation of Rip3 is likely an important target to inhibit cell necrosis and control the inflammatory response caused thereby. Studies have shown that protein phosphatase 1B (Ppm1b) can inhibit necroptosis in cultured cells and mice by dephosphorylating Rip3, and Ppm1b protein has become an important tool to control programmed necrosis. Given that programmed cell necrosis has been found to be closely related to the occurrence of inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases and other diseases, Ppm1b protein has shown great potential in the treatment of the above-mentioned diseases related to programmed cell necrosis.

[0213] 3.1 Construction of Expression Vectors for TINNeL-Ppm1b Complex and Other Control Protein

[0214] Expression vectors for recombinant protein containing TAT, INF7, protease cleavage site, multimerization domain, and cargo molecule Ppm1b (SEQ ID NO: 29) were constructed. The structural schematic diagram of each recombinant protein was shown in FIG. 13, the amino acid sequence of each component from the N-terminus to the C-terminus was shown in the table below. 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 and the overlap on upstream of the corresponding NdeI restriction site on pET-21b(+) were introduced at the 5′ end of the fragment by forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00005 TABLE 5 Components of delivery system-cargo molecule complex N-terminal .fwdarw. C-terminal components and sequences thereof pH- Protease Name of sensitive recognition Leucine complex CPP peptide sequence zipper Cargo Ppm1b None None None None Ppm1b T- Ppm1b Tat SEQ ID TI- Ppm1b INF7 NO: 29 TINNe- SEQ ID SEQ ID N + Ne Ppm1b NO: 14 NO: 12 TINNeL- SEQ ID Ppm1b NO: 1

[0215] 3.2 Expression and Purification of TINNeL-Ppm1b Protein and Other Control Proteins

[0216] 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 and inoculated into 5 ml of LB liquid medium containing ampicillin resistance and cultured overnight, and 1 ml of the bacterial liquid overnight cultured was taken and transferred to 500 ml of LB liquid medium containing ampicillin resistance, cultivated at 37° C. and 180 rpm until the bacterial liquid had an OD.sub.600 of about 0.6, and then inducer IPTG was added to a final concentration of 0.2 mM, and induction was performed at 25° C. for 8 hours; after the induction expression was completed, the cells were collected by centrifugation under 7000 g at 4° C. for 10 minutes, a part of the cells was taken to detect the induced expression of protein; then, the cells were resuspended with 10 ml of protein purification equilibration buffer (50 ml C.sub.3H.sub.8O.sub.3, 3.6342 g Tris(Hydroxymethyl)aminomethane was dissolved in 1 L double distilled water, and adjusted to pH 8.0), and disrupted by ultrasonication. Then, the supernatant was collected by centrifugation and loaded onto the Sulphopropyl (SP) cation exchange column of the AKTA protein purification system; then the equilibration buffer and high-salt eluent (50 ml C.sub.3H8O.sub.3, 116.88 g NaCl, 3.6342 g Tris(Hydroxymethyl)aminomethane was dissolved in 1 L of double-distilled water, and adjusted to pH 8.0) at different ratios were used to perform gradient elution to obtain the target protein. The protein concentration could be determined according to spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at −20° C.

[0217] 3.3 Effect of TINNeL-Ppm1b on TNF-α-Induced Necrosis of L292 Cells

[0218] L929 cells were inoculated into a 12-well cell culture plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 2*10.sup.6, the cells were rinsed three times with serum-free DMEM medium, and then the delivery system protein TINNeL-Ppm1b and other control proteins were added respectively in serum-free medium. After 3 hours of incubation, 1 ml of 20 ng/ml TNF-α and 20 mM z-VAD diluted with 10% FBS DMEM were added. After 10 hours of incubation, the cells were collected for PI staining. The ratio of cell necrosis in cells was observed by flow cytometry analysis. At the same time, lentivirus-transduced Ppm1b (Lenti-Ppm1b) and lentivirus (Lenti-vec) were used as controls, the lentiviruses with Ppm1b expression sequences and the control lentiviruses were packaged and collected on HEK-293T cells, and after infecting L929 cells for 24 h to make Ppm1b fully expressed in the cells, the infected L929 cells were re-plated for later TNF-α stimulation. The results were shown in FIG. 14. In the L929 treated with TINNeL-Ppm1b, the ratio of cell necrosis caused by TNF-α was the lowest, that was only about 10%, and its inhibitory effect was even better than that of the lentivirus infection group (20%) as a positive control.

[0219] At the same time, because Ppm1b inhibited cell necrosis by inhibiting the process of Rip3 phosphorylation, we tried to verify the contents of Rip3 and phosphorylated Rip3 (p-Rip3) in L929 cells in different treatment groups (by the same treatment method as above) by western blotting, so as to further determine that TINNe-Ppm1b inhibited the necrosis of TNF-α cells by inhibiting the phosphorylation of Rip3 more efficiently after entering the cytoplasm. The results were shown in FIG. 14, in which the contents of Rip3 in L929 cells of all treatment groups were basically the same, while for p-Rip3 in the phosphorylated state, the TINNeL-Ppm1b group showed a result significantly lower than those of other protein groups, which indicated that TINNeL-Ppm1b could more efficiently enter L929 cytoplasm and inhibit the necrosis caused by TNF-α by inhibiting the phosphorylation of Rip3.

[0220] 3.4 Evaluation of Inhibitory Activity of TINNeL-Ppm1b on TNF-α-Induced Systemic Inflammatory Response Syndrome

[0221] In mice, TNF-α-induced cell necrosis further triggers a systemic inflammatory response syndrome (SIRS) in mice and ultimately leads to mouse death. The TINNeL delivery system not only had high delivery efficiency, but also should be able to maintain a high delivery efficiency in the presence of serum, that was, it could still exert its delivery efficiency well in vivo. Therefore, we attempted to observe whether TINNeL-Ppm1b still had an ideal delivery efficiency in vivo based on TNF-α-induced SIRS model.

[0222] Through the tail vein route, 25 mmol of TINNeL-Ppm1b and other control proteins were injected into 6-week-old female BALB/C mice (12 per group), and 15 μg of TNF-α was also injected into the mice through the tail vein route 2 hours later, and the survival of the mice was observed every other hour thereafter (FIG. 15). The results showed that during the observation period (the survival rate was basically stable after 40 h), all the mice in the control group died of inflammation caused by TNF-α within 20 h, while the mice in the TINNeL-Ppm1b group were still 100% alive at the end of the observation period (FIG. 15). At the same time, TNF-α-induced SIRS could be directly observed from the degree of cecum injury in mice, so we injected 25 nmol of TINNeL-Ppm1b and other control proteins into 6-week-old female BALB/C mice (6 per group) through the tail vein route, 15 μg of TNF-α was also administered to the mice through the tail vein 2 hours later, and the mice were euthanized 10 hours later, and the cecum was removed for HE staining to observe the cecum damage of the mice in different treatment groups and the score of damage was given. The results showed that the mice in the TINNeL-Ppm1b group had almost uninjured cecum (FIG. 16), and the score was comparable to that of the Mock group (the control group that did not receive TNF-α), and could more effectively inhibit TNF-α-induced SIRS as compared with other proteins (FIG. 17). Therefore, based on the above results, we confirmed that TINNeL-Ppm1b could effectively inhibit TNF-α-induced SIRS in mouse, and TINNeL was a more effective delivery system.

Example 4: Application of TINNeL-Ppm1b in Treatment of Acute Liver Injury Caused by APAP

[0223] Acetaminophen (APAP) is a commonly used antipyretic and analgesic drug in clinical practice, but its overdose can lead to acute liver injury and the risk of 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 APAP-induced acute liver injury.

[0224] First, the mice aged 5 to 7 weeks (16-18 g) were injected with 500 mg/kg (non-lethal dose) of APAP through the tail vein route, and two injections of 25 nmol of TINNeL-Ppm1b and other control proteins prepared in Example 3 (7 mice in each group) were injected through the tail vein route 2 h and 6 h later, respectively; the blood sample was collected by orbital bleeding 6 h after APAP injection (12 h), the levels of ALT and AST in serum were detected, and the liver damage of the mice was evaluated. The results showed that the ALT and AST levels of the TINNeL-Ppm1b group were the lowest, which were basically the same as those of the APAP dilution buffer DMSO group (the increase of ALT/AST was caused by DMSO, and this part was not produced by the Rip3 pathway). Therefore, TINNeL-Ppm1b could achieve an ideal therapeutic effect of the APAP-induced acute liver injury (FIG. 18).

[0225] If the dose of APAP was further increased, it could lead to more severe liver damage and then induce the death of mice. Therefore, we tried to observe whether TINNeL-Ppm1b could treat the liver damage of mice at a lethal dose of APAP. Meanwhile, we used NAC, the only drug currently approved by FDA for the treatment of APAP-induced liver injury, as a control. First, BALB/C mice (16-18 g) aged 5 to 7 weeks were injected with 800 mg/kg (lethal dose) of APAP through the tail vein route, and then two injections of 25 mmol of TINNeL-Ppm1b, NAC, PBS (7 mice in each group) were injected through the tail vein route 2 h and 6 h later, respectively; and then the survival of mice was observed every other hour. The results were shown in FIG. 19. All the mice in the PBS group died within 18 h. During the observation period of 72 h (acute phase occurred within 24 h), the survival rate of the mice in the NAC group was 20%, while the TINNeL-Ppm1b group could reach 60%. Therefore, even for more severe acute liver injury caused by APAP, TINNeL-Ppm1b showed a good therapeutic effect, which was even better than the current clinical drug NAC. The above results fully demonstrated the advantages of TINNeL in delivery in vivo.

Example 5: Evaluation of Nucleic Acid Delivery Efficiency In Vitro and In Vivo of TINNeL-ZFP9

[0226] 5.1 Construction of Expression Vectors for TINNeL-ZFP9 and Other Control Protein

[0227] The expression vectors of recombinant protein containing TAT, INF7, protease cleavage site, multimerization domain, cargo molecule ZFP9 (SEQ ID NO: 31) were constructed, the structure of each recombinant protein was shown in FIG. 20, and the amino acid sequence of each component from the N-terminus to the C-terminus was shown in the following table. The construction method was as follows: the nucleic acid sequences encoding TAT, INF7, protease cleavage site, leucine zipper, ZFP9 were obtained by PCR amplification, and these parts were ligated by multiple rounds of PCR, and in the last round of PCR process, the NdeI restriction site and the overlap on upstream of the corresponding NdeI restriction site on pET-21b(+) were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pET-21b(+) were introduced at the 3′ end of the fragment through the reverse primer. The pET-21b(+) plasmid was subjected to double digestion with NdeI and BamHI. The insert fragments with overlaps were ligated to the digested vector pET-21b(+) by GIBSON assembly.

TABLE-US-00006 TABLE 6 Components of delivery system-cargo molecule complex N-terminal .fwdarw. C-terminal components and sequences thereof pH- Protease Name of Leucine sensitive recognition complex zipper CPP peptide sequence Cargo T-ZFP9 None Tat None None ZFP9- TI-ZFP9 SEQ ID INF7 NLS TINNe- NO: 14 SEQ ID N + Ne SEQ ID ZFP9 NO: 12 NO: 31 TINNeL- SEQ ID ZFP9 NO: 2

[0228] 5.2 Expression and Purification of TINNeL-ZFP9 and Control Proteins

[0229] 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 and inoculated into 5 ml of LB liquid medium containing ampicillin resistance and cultured overnight, and then 1 ml of the bacterial liquid overnight cultured was taken and transferred to 500 ml of LB liquid medium containing ampicillin resistance, cultivated at 37° C. and 180 rpm until the bacterial liquid had an OD.sub.600 of about 0.6, and then inducer IPTG was added to a final concentration of 0.2 mM, and induction was performed at 25° C. for 8 hours; after the induction expression was completed, the cells were collected by centrifugation under 7000 g at 4° C. for 10 minutes, and a part of the cells was taken to detect the induced expression of protein; then, the cells were resuspended with 10 ml of protein purification equilibration buffer (50 ml C.sub.3H.sub.8O.sub.3, 3.6342 g Tris(Hydroxymethyl)aminomethane was dissolved in 1 L double distilled water, and adjusted to pH 8.0), and disrupted by ultrasonication. Then the supernatant was collected by centrifugation and loaded onto the Sulphopropyl (SP) cation exchange column of the AKTA protein purification system; then the equilibration buffer and high-salt eluent (50 ml C.sub.3H.sub.8O.sub.3, 116.88 g NaCl, 3.6342 g Tris(hydroxymethyl)aminomethane was dissolved in 1 L of double-distilled water, and adjusted to pH 8.0) at different ratios were used to perform gradient elution to obtain the target protein. The protein concentration could be determined according to spectrophotometer or BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at −20° C.

[0230] 5.3 Construction of Eukaryotic Expression Plasmid Containing ZFP9 Binding Site

[0231] The expression vector (pTT5-tdTomato-6BS plasmid) containing tdTomato coding sequence and ZFP9 binding site was constructed, and its schematic structure was shown in FIG. 21. The tdTomato coding sequence (SEQ ID NO: 33) and the sequence of ZFP9 binding sites (6*binding sites) (SEQ ID NO: 34) were obtained by amplification through PCR, and the two were ligated by two rounds of PCR, and during the second round of PCR, the Hind III restriction site and the overlap on upstream of the corresponding Hind III restriction site on pTT5 vector were introduced at the 5′ end of the fragment through the forward primer, and the BamHI restriction site and the overlap on downstream of the corresponding BamHI restriction site on pTT5 vector were introduced at the 3′ end of the fragment through the reverse primer. The pTT5 plasmid was double-digested with Hind III and BamH I. The insert fragments with overlaps were ligated to the digested pTT5 vector by GIBSON assembly.

[0232] 5.4 Evaluation of Delivery Efficiency of TINNeL-ZFP9 in Delivery of Plasmid DNA

[0233] First, HEK-293T cells were inoculated into a 12-well plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 5*10.sup.6; 5 μM of the delivery system protein or other control proteins and 5 g pTT5-tdTomato-6BS plasmid were co-incubated at 37° C. for 30 min to fully form a complex; the cells were rinsed three times with serum-free DMEM medium, and then the complex was added and incubated for 3 h; the medium was replaced with 10% FBS DMEM and the culturing was continued, and the red fluorescent protein expression was observed by flow cytometry analysis after the medium was replaced for 12 h, 24 h, and 36 h respectively. The results were shown in FIG. 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 at delivering pTT5-tdTomato-6BS attached to it into the nucleus, thereby realizing the expression of the red fluorescent gene on the plasmid.

[0234] 5.5 Evaluation of Delivery Efficiency of TINNeL-ZFP9 Under Serum Conditions

[0235] In this example, the transfection effect of TINNeL-ZFP9 under serum conditions was investigated, and the commercial transfection reagent X-tremeGENE, which could work under serum conditions, was used as a control.

[0236] First, HEK-293T cells were inoculated into a 12-well plate and cultured overnight. Before protein treatment, it was ensured that the number of cells in each well was about 5*10.sup.6; 5 μM of TINNeL-ZFP9 and 5 μg pTTS-tdTomato-6BS plasmid was co-incubated at 37° C. for 30 min to fully form a complex; the complex was added under 100% FBS serum conditions and serum-free conditions and incubated for 3 hours; the medium was replaced with 10% FBS DMEM and the culturing 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 X-tremeGENE, then allowed to stand at room temperature for 30 min, the incubation was performed under 100% FBS serum conditions and serum free conditions for 8 h, and then the medium was replaced with 10% FBS DMEM and the culturing was continued. For the above treatment groups, the expression of red fluorescent protein was observed by flow cytometry analysis after the medium was replaced for 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, and 84 h, respectively. The results were shown in FIG. 23. Under serum-free conditions, the proportion of red fluorescent cells in the TINNeL-ZFP9 group increased faster, but after reaching the plateau phase, the proportion was basically the same as that in the X-tremeGENE group. However, under the condition of 100% FBS, there was a difference of nearly 10% in the proportion of red fluorescent cells at each time point, indicating that TINNeL-ZFP9 showed a more efficient transfection ability under serum conditions.

[0237] 5.5 Evaluation of Delivery Efficiency of TINNeL-ZFP9 in Mice

[0238] The pTT5-Luc-6BS expression plasmid with Luciferase encoding gene (SEQ ID NO:35) and ZFP9 binding site (SEQ ID NO:34) was constructed, and the construction method was the same as the above 5.3. After 4 nmol of TINNeL-ZFP9 or other control proteins TINNe-ZFP9, T-ZFP9 were mixed with 5 μg pTT5-Luc-6BS expression plasmid for 30 minutes, it was injected into the left leg muscle of nude mice, and the fluorescence intensity was imaged and analyzed 24 hours later. The results were shown in FIG. 24. The T-ZFP9 and no-treatment groups had almost no fluorescence detected, while the TINNe-ZFP9 had weak fluorescence, while the TINNeL-ZFP9 group had strong fluorescence detected. Therefore, it was confirmed in mouse that TINNeL-ZFP9 could efficiently bind and deliver DNA.

[0239] Although specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention. The full division of the present invention is given by the appended claims and any equivalents thereof