Complex for intracellular delivery of molecules

Abstract

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

Description

Technical Field

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

[0002] The cell membrane, as a selectively permeable barrier, is crucial for cell survival and function. Although small molecules can cross the cell membrane through natural processes of the cell or direct diffusion through the lipid bilayer, in most cases, the efficient passage of exogenous active biomacromolecules and other intracellular cargos across the cytoplasmic membrane remains the main obstacle in the process of cell delivery. Therefore, a molecular transport tool that can effectively improve the transport efficiency of intracellular cargos to living cells is extremely important for its applications in the fields of biomedicine and the like.

[0003] Cell-penetrating peptides (CPPs) are currently one of the most common and effective carriers for realizing the cell uptake process. Cell-penetrating peptides, usually containing 5-30 amino acids, can carry biomacromolecules across the cell membrane into the cell by means of chemical cross-linking, fusion expression or non-covalent binding. So far, hundreds of CPPs derived from natural proteins or artificially synthesized have been reported and used in the research of intracellular delivery of intracellular cargos. According to their chemical properties, they can be divided into three categories: (1) cationic type, rich in arginine and lysine residues, with strong positive charges at physiological pH; (2) amphiphilic type, containing polar and non-polar regions. In addition to lysine and arginine distributed throughout the sequence, they are also rich in hydrophobic residues such as valine, leucine, isoleucine and alanine, etc.; (3) hydrophobic type, mainly containing non-polar amino acids. CPPs interact with the negative charges on the cell membrane surface or the hydrophobic lipid bilayer through their strong positive charges or hydrophobic groups. When carrying small molecules, they can directly translocate across the cell membrane in a non-energy-consuming manner. When carrying biomacromolecules, they basically enter the cell by energy-dependent endocytosis. Due to the advantages of low dosage, short transport time, controllable dose, simple operation and low toxicity and side effects of CPPs when mediating endocytosis, CPPs have been widely used in vitro and in vivo to transport polypeptides, proteins, oligonucleotides, plasmids, liposomes, metal ions, small molecule fluorescence, nanoparticles and so on.

[0004] Although CPPs have many advantages in transducing biomacromolecules, there are still some limitations, among which the first problem to be solved is their low transmembrane delivery efficiency. It is generally believed that when transducing the internalized cargo into cells, CPPs interact with the cell membrane surface and then enter the cells through endocytosis. Endocytosis can be mainly divided into four ways according to different endocytic mechanisms: macropinocytosis, clathrin-mediated endocytosis, caveolae / lipid raft-mediated endocytosis, and clathrin / caveolae-independent endocytosis. These different endocytic ways will ultimately generate endosomes, which mature unidirectionally from early endosomes to late endosomes with a gradual decrease in the pH inside the vesicles, and finally fuse with lysosomes. The internalized cargo in cells needs to escape from endosomes into the cytoplasm before entering lysosomes; otherwise, it will ultimately enter lysosomes and be degraded without being able to function. Existing studies have shown that only 1% of the biomacromolecules carried by CPPs can successfully escape from endosomes, and most of them ultimately end up in lysosomes and are degraded. Therefore, the endosomal escape of the internalized cargo is the key limiting factor in the intracellular delivery process of CPPs, and its efficiency determines the overall delivery efficiency.

[0005] For improving the escape efficiency of vesicles, the main strategy is to disrupt the integrity of the vesicle membrane during the process of maturation and acidification, so that its content (including the intracellular cargo to be delivered) is released into the cytoplasm. It has been reported that the addition of buffers such as chloroquine, methylamine, or ammonium chloride to the system can physically promote the osmotic rupture of endocytic vesicles, but their strong cytotoxicity hinders their clinical application. Currently, the most effective way is to use pH-sensitive fusogenic peptides derived from viruses, bacteria, animals, plants, or humans. pH-sensitive peptides contain a certain proportion of hydrophobic amino acids and undergo a drastic conformational change at low pH values. After endocytosis mediated by CPPs, during the process of maturation and acidification of endocytic vesicles, once the pH value drops to the critical point, the pH-sensitive peptide conjugated to CPPs undergoes a conformational change and binds to the lipid bilayer of the vesicle membrane, thus severely disturbing the integrity of the phospholipid bilayer membrane, forming small pores or causing membrane lysis on it, and finally releasing the biomacromolecules to be transported into the cytoplasm. Currently, the commonly used pH-sensitive peptides are a polypeptide in the stem region of the influenza virus hemagglutinin antigen (hereinafter referred to as HA2), and INF7 with lower toxicity and better membrane-breaking effect after artificial modification based on HA2. After fusion expression with CPPs and protein molecules, they can improve the escape efficiency of endocytic vesicles of transport protein molecules. However, although the escape efficiency of endocytic vesicles of the CPPs macromolecular intracellular delivery system after fusion with pH-sensitive peptides is indeed improved, a considerable part of the transported macromolecules still remain in the endocytic vesicles (obvious punctate distribution can be seen with fluorescent protein as the transported macromolecule).

[0006] Therefore, there is still a need to develop new delivery systems to achieve efficient release of the transported biomacromolecules from endocytic vesicles. Summary of the Invention

[0007] Through a large number of experiments and repeated explorations, the inventors of the present application unexpectedly found that the combination of pH-sensitive peptides and specific protease recognition sequences as delivery carriers can significantly improve the release of cargo molecules from endocytic vesicles, thereby significantly enhancing the cytoplasmic delivery efficiency of cargo molecules and enabling cargo molecules to fully exert their corresponding biological functions. Based on these findings, the inventors developed a vector system capable of achieving efficient cytoplasmic delivery.

[0008] Fusion protein

[0009] Accordingly, a first aspect of the present invention provides a fusion protein comprising a cell-penetrating peptide, a pH-sensitive fusogenic peptide, and a protease recognition sequence, wherein the protease is selected from furin and / or lysosomal cysteine protease.

[0010] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: R-X1-X2-R ↓ (SEQ ID NO:1), wherein X1 is any amino acid, X2 is K or R, and ↓ represents the cleavage site.

[0011] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: R-R-X1-X2-R ↓ (SEQ ID NO:2).

[0012] 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), asparagine (N), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

[0013] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: RRHKR ↓ (SEQ ID NO:3).

[0014] In certain embodiments, the furin recognition sequence comprises or consists of the following sequence: QSVASSRRHKR ↓ FAGV(SEQ ID NO:4).

[0015] In certain embodiments, the lysosomal cysteine protease is selected from cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D, or cathepsin H.

[0016] In certain embodiments, the lysosomal cysteine protease is cathepsin L.

[0017] In certain embodiments, the cathepsin L recognition sequence comprises or consists of the following sequence: NNTHDLVGDVRLAGV(SEQ ID NO:6).

[0018] In certain embodiments, the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence. In certain embodiments, the protease recognition sequence is a single-chain polypeptide that comprises, from the N-terminus to the C-terminus, a furin recognition sequence and a cathepsin L recognition sequence, or that comprises, from the N-terminus to the C-terminus, a cathepsin L recognition sequence and a furin recognition sequence.

[0019] In certain embodiments, the protease recognition sequence comprises RRHKR (SEQ ID NO:3) and NNTHDLVGDVRLAGV (SEQ ID NO:6). In certain embodiments, the protease recognition sequence comprises SEQ ID NO:4 and SEQ ID NO:6.

[0020] In the present invention, the term "pH-sensitive fusogenic peptide" is used interchangeably with "pH-sensitive peptide" and refers to a class of polypeptides that are capable of undergoing a conformational change under acidic conditions (e.g., pH < 6.5) to promote fusion with the endocytic vesicle membrane. When the pH-sensitive peptide is endocytosed by a cell, during the maturation and acidification of the endocytic vesicle, once the pH value drops to the critical point, such polypeptides undergo a conformational change and bind to the lipid bilayer of the vesicle membrane, thereby severely perturbing the integrity of the phospholipid bilayer membrane, forming small pores or causing vesicle membrane lysis thereon, and thus releasing the biomacromolecules to be transported 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 hereby incorporated by reference in their entirety.

[0021] The pH-sensitive peptides that can be used in the fusion proteins of the present invention can be selected from the following proteins or polypeptides, or be derived from the following proteins or polypeptides:

[0022] Viral protein sources: HA2 (influenza virus) and its mutants KALA, GALA; penton base (adenovirus or rhinovirus), gp41 (HIV), L2 (papillomavirus), envelope protein (West Nile virus);

[0023] Bacterial protein sources: Listeriolysin O (LLO), Pneumococcal pneumolysin (PLO), Streptococcal streptolysin O (SLO), Diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Shiga toxin, Cholera toxin;

[0024] Plant protein sources: Ricin, Saporin, Gelonin toxin;

[0025] Human / animal protein sources: Human calcitonin, fibroblast growth factor receptor, Melittin;

[0026] Synthetic polypeptides: (R-Ahx-R)(4)AhxB, Penetratin (pAntp), EB1, Bovine prion protein (bPrPp), Sweet Arrow Peptide (SAP), Poly(L-histidine), Proline-rich.

[0027] In certain embodiments, the pH-sensitive fusion peptide is selected from influenza virus HA2 (SEQ ID NO:38) or a mutant thereof, Melittin (SEQ ID NO:41), and any combination thereof. In certain embodiments, the mutant of influenza virus HA2 is selected from INF7 (SEQ ID NO:8), KALA (SEQ ID NO:39), or GALA (SEQ ID NO:40).

[0028] In certain embodiments, the pH-sensitive fusion peptide comprises INF7. In certain embodiments, the pH-sensitive fusion peptide comprises the following sequence or consists of: SEQ ID NO:8.

[0029] In the present invention, the term "cell penetrating peptide (CPP)" is also known as "cell-penetrating peptide", "protein translocation domain (PTD)", "Trojan horse peptides", or "transduction peptide", etc. It refers to a polypeptide that can promote the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids). Such polypeptides are well-known in the art and are described, for example, in Stewart KM, et al. Org Biomol Chem. 2008 Jul 7;6(13):2242-55 and Chinese Patent Application CN101490081A (which are all incorporated herein by reference in their entirety); or can be obtained by methods known in the art, such as the method described in detail in US Patent Application US 2008 / 0234183, which is incorporated herein by reference in its entirety.

[0030] CPPs that can be used for the fusion proteins of the present invention include, but are not limited to: cationic type: Penetratin, HIV-TAT-47-57, HIV-1Rev 34–50, FHV coat-35-49, Oligoarginines (R9–R12), CCMV Gag-7-25, S413-PV, VP22, BP16, DPV3, DPV6, FAH coat protein, Protamine 1, human cJun, Engrailed-2, Islet-1, HoxA-13, TP10, etc.; amphiphilic type: transportan, Transportan 10, Pep-1, MPGα, MPGβ, CADY, Pepfect6, Pepfect14, Pepfect15, NickFect, Hel, sC18, pVEC, ARF(1-22), YTA2, PAR1 (Palmitoyl-SFLLRN), F2Pal10 (Palmitoyl-SFLLRN), BPrPp(1-30), hLF peptide (19–40), Buforin 2, Crotamine, Azurin p18, hCT peptide (18-32), S413-PVrev, etc.; hydrophobic type: Kaposi's sarcoma fibroblast growth factor, signal peptide derived from the Ig light chain of Caiiman crocodylus, integrin β3 fragment, Grb2-SH2 domain, HIV-1gp41(1-23), HBV translocation motif, sperm-egg fusion protein (89–111), human calcitonin (9–32), Pep-7, C105Y, K-FGF, etc.

[0031] In addition, the CPP used in the fusion protein of the present invention can also be selected from polypeptide sequences having about 60, 70, 80, 90, 95, 99% or 100% sequence identity with any of the polypeptide sequences described above, as long as the polypeptide sequence still retains its biological activity, that is, promotes the cellular uptake of molecules.

[0032] In certain embodiments, the cell-penetrating peptide is selected from Penetratin (SEQ ID NO: 42), Tat-derived peptides, Rev(34-50) (SEQ ID NO: 44), VP22 (SEQ ID NO: 45), transportan (SEQ ID NO: 46), Pep-1 (SEQ ID NO: 47), Pep-7 (SEQ ID NO: 48), and any combination thereof. In certain embodiments, the Tat-derived peptide is selected from Tat(48-60) (SEQ ID NO: 10) or Tat(47-57) (SEQ ID NO: 43).

[0033] In certain embodiments, the cell-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60). In certain embodiments, the cell-penetrating peptide comprises or consists of the following sequence: SEQ ID NO: 10.

[0034] In some embodiments, the fusion protein of the present invention comprises the pH-sensitive fusion peptide, the cell-penetrating peptide, and the protease recognition sequence from the N-terminus to the C-terminus. In certain embodiments, the fusion protein comprises the pH-sensitive fusion peptide, the cell-penetrating peptide, the furin recognition sequence, and the cathepsin L recognition sequence from the N-terminus to the C-terminus. In certain embodiments, the fusion protein comprises the pH-sensitive fusion peptide, the cell-penetrating peptide, the cathepsin L recognition sequence, and the furin recognition sequence from the N-terminus to the C-terminus.

[0035] In other embodiments, the fusion protein of the present invention comprises the cell-penetrating peptide, the pH-sensitive fusion peptide, and the protease recognition sequence from the N-terminus to the C-terminus. In certain embodiments, the fusion protein comprises the cell-penetrating peptide, the pH-sensitive fusion peptide, the furin recognition sequence, and the cathepsin L recognition sequence from the N-terminus to the C-terminus. In certain embodiments, the fusion protein comprises the cell-penetrating peptide, the pH-sensitive fusion peptide, the cathepsin L recognition sequence, and the furin recognition sequence from the N-terminus to the C-terminus.

[0036] In certain exemplary embodiments, the fusion protein comprises or consists of a sequence selected from any one of the sequences shown in SEQ ID NOs: 12-14.

[0037] In certain embodiments, the fusion protein may further comprise a protein tag at its N-terminus. In certain embodiments, the protein tag has a solubilizing effect. In certain embodiments, the protein tag is selected from TrxA, SUMO, NusA, MBP, GST.

[0038] In a second aspect of the present invention, there is also provided a fusion protein which further comprises a specific binding sequence on the basis of the fusion protein described in the first aspect, and the specific binding sequence allows another molecule (such as a polypeptide, a protein or a nucleic acid (such as DNA)) to specifically bind thereto. As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody (or its antigen-binding fragment) and the antigen (or antigenic epitope) it targets, or the reaction of forming a heterodimer between two amino acid sequences (such as two leucine zipper domains in an antiparallel manner).

[0039] In certain embodiments, the specific binding sequence comprises a leucine zipper, and the leucine zipper is capable of forming a heterodimer with its reverse sequence. In certain embodiments, the specific binding sequence comprises leucine zipper NZ or CZ. It is known in the art that leucine zipper NZ and CZ are reverse sequences of each other and have a strong interaction force therebetween, so that the antiparallel leucine zipper NZ and CZ can form a heterodimer. In certain embodiments, the leucine zipper NZ comprises the sequence shown in SEQ ID NO:49. In certain embodiments, the leucine zipper CZ comprises the sequence shown in SEQ ID NO:50.

[0040] In some embodiments, the specific binding sequence comprises the sequence shown in SEQ ID NO:49. In other embodiments, the specific binding sequence comprises the sequence shown in SEQ ID NO:50.

[0041] In certain embodiments, the specific binding sequence is located at the C-terminus of the protease recognition sequence. In certain embodiments, the fusion protein comprises the specific binding sequence at its C-terminus.

[0042] In certain embodiments, the fusion protein may further comprise a protein tag at its N-terminus. In certain embodiments, the protein tag has a solubilizing effect. In certain embodiments, the protein tag is selected from TrxA, SUMO, NusA, MBP, GST.

[0043] Preparation of fusion protein

[0044] The fusion protein of the present invention can be prepared by various methods known in the art. For example, it can be produced by genetic engineering methods (recombinant techniques), or can also be produced by chemical synthesis methods (such as Fmoc solid-phase methods). The fusion protein of the present invention is not limited by its production method.

[0045] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule which comprises a nucleotide sequence encoding the fusion protein of the first or second aspect of the present invention.

[0046] In another aspect, the present invention provides a vector (such as a cloning vector or an expression vector) that contains the isolated nucleic acid molecule as described above. In certain embodiments, the vector is, for example, a plasmid, cosmid, phage, etc.

[0047] In another aspect, the present invention provides a host cell that contains the isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, for example, mouse cells, human cells, etc.).

[0048] In another aspect, a method for preparing the fusion protein of the first or second aspect of the present invention is provided, which includes culturing the host cell as described above under conditions that allow the expression of the fusion protein, and recovering the fusion protein from the cultured host cell culture.

[0049] Complex

[0050] In another aspect, the present invention provides a complex that contains the fusion protein of the first or second aspect of the present invention and a cargo molecule. The cargo molecule can be any molecule.

[0051] In certain embodiments, the cargo molecule is selected from nucleic acids, peptides or proteins, saccharides, lipids, chemical compounds, and any mixture thereof.

[0052] In certain embodiments, the nucleic acid is selected from DNA molecules, RNA molecules, siRNA, antisense oligonucleotides, ribozymes, aptamers, and any combination thereof.

[0053] In certain embodiments, the molecular weight of the cargo molecule is less than 10,000 Da, for example, less than 5,000 Da, less than 3,000 Da, or less than 1,000 Da.

[0054] In certain embodiments, the cargo molecule contains a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a chemiluminescent substance, or biotin, etc.

[0055] In certain embodiments, the cargo molecule contains an epitope tag, a reporter gene sequence, and / or a nuclear localization signal (NLS) sequence. In certain embodiments, the cargo molecule is a peptide or protein.

[0056] Epitope tags that can be used in cargo molecules are well-known to those skilled in the art, and examples thereof include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select a suitable epitope tag according to the desired purpose (e.g., purification, detection or tracing). In certain exemplary embodiments, the cargo molecule comprises a His tag.

[0057] Reporter gene sequences that can be used in cargo molecules are well-known to those skilled in the art, and examples thereof include, but are not limited to, GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.

[0058] Nuclear localization signal (NLS) sequences that can be used in cargo molecules are well-known to those skilled in the art, and examples thereof include, but are not limited to, the NLS of the large T antigen of SV40 virus. In certain exemplary embodiments, the NLS sequence is as shown in SEQ ID NO:15.

[0059] In some embodiments, the fusion protein of the present invention is fused to a cargo molecule, wherein the cargo molecule is a peptide or a protein. In certain embodiments, the fusion protein is as defined in the first aspect.

[0060] In certain embodiments, the cargo molecule is fused to the N-terminus or C-terminus of the fusion protein. In certain embodiments, the cargo molecule is fused to the C-terminus of the fusion protein.

[0061] In some embodiments, the complex of the present invention comprises a single-chain polypeptide, which from the N-terminus to the C-terminus comprises: the pH-sensitive fusion peptide, the cell-penetrating peptide, the protease recognition sequence, and the cargo molecule. In certain embodiments, the single-chain polypeptide from the N-terminus to the C-terminus comprises the pH-sensitive fusion peptide, the cell-penetrating peptide, the furin protease recognition sequence, the cathepsin L recognition sequence, and the cargo molecule. In certain embodiments, the single-chain polypeptide from the N-terminus to the C-terminus comprises the pH-sensitive fusion peptide, the cell-penetrating peptide, the cathepsin L recognition sequence, the furin protease recognition sequence, and the cargo molecule.

[0062] In other embodiments, the complex of the present invention comprises a single-chain polypeptide, which from the N-terminus to the C-terminus comprises: the cell-penetrating peptide, the pH-sensitive fusion peptide, the protease recognition sequence, and the cargo molecule. In certain embodiments, the single-chain polypeptide from the N-terminus to the C-terminus comprises the cell-penetrating peptide, the pH-sensitive fusion peptide, the furin protease recognition sequence, the cathepsin L recognition sequence, and the cargo molecule. In certain embodiments, the single-chain polypeptide from the N-terminus to the C-terminus comprises the cell-penetrating peptide, the pH-sensitive fusion peptide, the cathepsin L recognition sequence, the furin protease recognition sequence, and the cargo molecule.

[0063] In certain exemplary embodiments, the cargo molecule is a zinc finger protein (e.g., ZFP9), a protein phosphatase (e.g., Ppm1b), or a Cas effector protein (e.g., Cas9). The expression "Cas effector protein" refers to the effector protein of the CRISPR-Cas system. In certain exemplary embodiments, the zinc finger protein or Cas effector protein comprises an NLS sequence.

[0064] In other embodiments, the fusion protein of the present invention is chemically coupled to the cargo molecule. The "chemical coupling" refers to the bonding obtained in a chemical reaction between the reactive groups contained in the fusion protein and the reactive groups contained in the cargo molecule, and the two parts are connected by a covalent bond after the chemical reaction. Before the above chemical reaction (coupling reaction), the fusion protein, the cargo molecule, or both can be modified with a linker molecule in an independent reaction so that they respectively contain the reactive groups required for the chemical coupling. The choice of the linker molecule used to modify the fusion protein or the cargo molecule depends on the coupling strategy used. In certain embodiments, the fusion protein is as defined in the first aspect.

[0065] 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.

[0066] In certain embodiments, the cargo molecule is coupled to the N-terminus or C-terminus of the fusion protein. In certain embodiments, the cargo molecule is coupled to the C-terminus of the fusion protein.

[0067] In certain embodiments, the cargo molecule is a nucleic acid.

[0068] In other embodiments, the fusion protein of the present invention is non-covalently linked to the cargo molecule.

[0069] In certain embodiments, the fusion protein is conjugated to the cargo molecule by electrostatic interaction. In certain embodiments, the cargo molecule is a nucleic acid.

[0070] In certain embodiments, the fusion protein is as defined in the second aspect, and the fusion protein is non-covalently linked to the cargo molecule through a specific binding sequence contained therein. In such embodiments, the cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein.

[0071] In certain embodiments, the domain capable of specifically binding to the specific binding sequence in the fusion protein is an amino acid sequence.

[0072] In certain embodiments, the cargo molecule is a peptide or a protein. In certain embodiments, the cargo molecule contains, at its N-terminus, the amino acid sequence capable of specifically binding to the specific binding sequence in the fusion protein.

[0073] In certain embodiments, the specific binding sequence in the fusion protein contains a leucine zipper, and the cargo molecule contains the reverse sequence of the leucine zipper, such that the leucine zipper can form a heterodimer with this reverse sequence.

[0074] In certain embodiments, the specific binding sequence in the fusion protein contains leucine zipper NZ (e.g., the sequence shown in SEQ ID NO:49), and the cargo molecule contains leucine zipper CZ (e.g., the sequence shown in SEQ ID NO:50).

[0075] In certain embodiments, the specific binding sequence in the fusion protein contains leucine zipper CZ (e.g., the sequence shown in SEQ ID NO:50), and the cargo molecule contains leucine zipper NZ (e.g., the sequence shown in SEQ ID NO:49).

[0076] Preparation of complex

[0077] The complex of the present invention can be prepared by various methods known in the art. For example, it can be produced by genetic engineering methods (recombinant techniques), or can also be produced by chemical synthesis methods (such as the Fmoc solid-phase method). The complex of the present invention is not limited by the manner of its production.

[0078] In some embodiments, when the complex contains a fusion protein fused with a cargo molecule, the complex of the present invention can be obtained by genetic engineering recombinant techniques. For example, a DNA molecule encoding the complex is obtained by chemical synthesis or PCR amplification. The obtained DNA molecule is inserted into an expression vector, and then the host cell is transfected. Then, the transfected host cell is cultured under specific conditions, and the complex of the present invention is expressed.

[0079] Thus, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the complex as described above.

[0080] In another aspect, the present invention provides a vector (such as a cloning vector or an expression vector) comprising the isolated nucleic acid molecule as described above. In certain embodiments, the vector is, for example, a plasmid, a cosmid, a phage, etc.

[0081] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as Escherichia coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, for example, mouse cells, human cells, etc.).

[0082] In another aspect, a method for preparing the complex as described above is provided, which comprises culturing the host cell as described above under conditions allowing the expression of the complex, and recovering the complex from the cultured host cell culture.

[0083] In other embodiments, when the complex comprises a chemically conjugated fusion protein and a cargo molecule, the complex of the present invention can be obtained by the following exemplary method: mixing the fusion protein and the cargo molecule under conditions allowing a chemical reaction to occur between the reactive groups respectively comprised in the fusion protein and the cargo molecule, so that the two moieties are linked by a covalent bond. In certain embodiments, the method further comprises: modifying the fusion protein, the cargo molecule, or both, with a linker molecule so that they respectively comprise the reactive groups required for the above chemical reaction. In certain embodiments, the cargo molecule is a nucleic acid.

[0084] In other embodiments, when the complex comprises a fusion protein 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 fusion protein of the present invention and the cargo molecule to form a mixture; and (2) incubating the mixture so that the fusion protein and the cargo molecule form a complex. In certain embodiments, the cargo molecule is a nucleic acid.

[0085] In other embodiments, when the complex comprises a fusion protein and a cargo molecule that are specifically bound but not covalently linked, the complex of the present invention can be obtained by the following exemplary method: (1) mixing the fusion protein described in the second aspect of the present invention and the cargo molecule, the cargo molecule comprising a domain that specifically binds to the specific binding sequence in the fusion protein; and (2) incubating the mixture so that the fusion protein and the cargo molecule form a complex. In certain embodiments, the cargo molecule is a polypeptide or a protein.

[0086] Composition

[0087] When the fusion protein of the present invention can be linked to a cargo molecule by non-covalent interaction, a delivery complex can be obtained by mixing the fusion protein and the cargo molecule. Thus, in another aspect, the present invention further provides a composition comprising the fusion protein of the present invention and a cargo molecule.

[0088] In certain embodiments, the cargo molecule is selected from nucleic acids, peptides or proteins, saccharides, lipids, chemical compounds, and any mixtures thereof. In certain embodiments, the nucleic acid is selected from DNA molecules, RNA molecules, siRNA, antisense oligonucleotides, ribozymes, aptamers, and any combination thereof.

[0089] In certain embodiments, the cargo molecule is selected from nucleic acids.

[0090] In certain embodiments, the cargo molecule is a polypeptide or a protein.

[0091] In certain embodiments, the fusion protein is as defined in the second aspect. The cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein.

[0092] Use and method

[0093] The fusion protein of the present invention can efficiently release the cargo molecule from the endocytic vesicle, and once the cargo molecule is available in the cytoplasm, they can exert any function associated therewith. Therefore, the fusion protein of the present invention can be used as an intracellular delivery reagent and thus further used in research as well as therapeutic and diagnostic applications.

[0094] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the fusion protein, complex, composition, isolated nucleic acid molecule, vector or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0095] In certain embodiments, the cargo molecule comprised in the complex or composition is a pharmaceutically active agent.

[0096] In certain embodiments, the cargo molecule comprised in the complex or composition is a detectable label. The label can be used for diagnosis, for studying drug disposition (e.g., absorption, distribution, metabolism, excretion), for studying the efficacy or side effects of a treatment or a drug, etc.

[0097] In another aspect, the present invention also relates to the use of the fusion protein, isolated nucleic acid molecule, vector or host cell of the present invention comprising a nucleotide sequence encoding the fusion protein, as a delivery reagent (e.g., intracellular delivery reagent and / or transfection reagent) for the preparation of a drug.

[0098] In another aspect, the present invention also relates to the use of the complex or composition of the present invention, or an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding the complex or composition, in the preparation of a drug for treating a disease; wherein, the cargo molecule comprised in the complex or composition is capable of treating the disease.

[0099] In certain embodiments, the disease is a disease associated with necroptosis, wherein the cargo molecule comprises protein phosphatase 1B. In certain embodiments, the diseases associated with necroptosis include liver injury (such as drug-induced liver injury), inflammatory diseases, ischemia-reperfusion injury, and / or neurodegenerative diseases.

[0100] The fusion protein, complex or composition, or pharmaceutical composition of the present invention can be in any form known in the medical field. For example, it can be in the form of tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, lyophilized powders), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use.

[0101] The fusion protein, complex or composition, or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to oral, rectal, parenteral or topical administration.

[0102] An exemplary route of administration is oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, etc. In addition to the active compound, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl acetate, acetic acid ethyl ester, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan and their mixtures. In addition to the inert diluent, liquid dosage forms for oral administration may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweetening agents, flavoring agents and aromatic agents, etc. Solid dosage forms for oral administration include capsules, tablets, pills, lozenges, powders, granules, etc. In addition to the active compound, solid dosage forms may contain pharmaceutically acceptable inert excipients or carriers, such as fillers (such as lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, crospovidone and calcium sulfate); binders (such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic); wetting agents (such as cetyl alcohol and glyceryl monostearate); disintegrants (such as agar, calcium carbonate, starch, alginic acid, sodium carboxymethyl cellulose, sodium carboxymethyl starch); lubricants (such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate); and their mixtures.

[0103] The fusion protein, complex or composition, or pharmaceutical composition of the present invention can also be administered by non-oral routes.

[0104] Thus, another exemplary route of administration is parenteral administration, for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and infusion. Dosage forms for parenteral administration can be injection preparations, including injection solutions, sterile powders for injection, or concentrated solutions for injection. In addition to the active compound, injection dosage forms may contain pharmaceutically acceptable carriers such as sterile water, Ringer's solution, and isotonic sodium chloride solution, and appropriate additives such as antioxidants, buffers, and bacteriostatic agents may also be added according to the nature of the drug.

[0105] Another exemplary route of administration is topical administration, for example, transdermal administration (such as administration through a transdermal patch or iontophoresis 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 compound, topical dosage forms may contain components that can enhance the absorption or penetration of the active compound through the skin or other regions of action.

[0106] Another exemplary route of administration is rectal administration. Dosage forms for rectal administration can be suppositories.

[0107] In addition, other carrier materials and administration methods known in the pharmaceutical field can also be used. The fusion proteins, complexes, or compositions, or pharmaceutical compositions of the present invention can be prepared by any well-known pharmaceutical processes, such as effective formulation and administration methods.

[0108] In another aspect, the present invention provides a kit that contains the fusion protein, complex, composition, isolated nucleic acid molecule, vector, or host cell of the present invention. In certain embodiments, the kit further includes instructions for transfection and / or intracellular delivery. In certain embodiments, the kit is used for transfection and / or intracellular delivery of cargo molecules (for example, nucleic acids, peptides, or proteins, saccharides, lipids, chemical compounds, and any mixtures thereof). In certain embodiments, the cells are mammalian cells, such as human cells.

[0109] In another aspect, the present invention also relates to the use of the fusion protein, complex, composition, isolated nucleic acid molecule, vector, or host cell of the present invention as a delivery reagent (such as a transfection reagent or an intracellular delivery reagent). In certain embodiments, the delivery reagent is used for intracellular delivery of cargo molecules (for example, nucleic acids, peptides, or proteins, saccharides, lipids, chemical compounds, and any mixtures thereof). In certain embodiments, the cells are mammalian cells, such as human cells.

[0110] In another aspect, the present invention provides a method for delivering a molecule into a cell, which includes contacting the cell with a complex of the present invention, wherein the complex includes the molecule.

[0111] In certain embodiments, the contacting of the cells with the complex is performed in vivo.

[0112] In certain embodiments, the contacting of the cells with the complex is performed ex vivo.

[0113] In certain embodiments, the contacting of the cells with the complex is performed in vitro.

[0114] In certain embodiments, the cells are eukaryotic cells, such as mammalian cells, such as human cells.

[0115] Term definition

[0116] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the operation steps such as cell culture, biochemistry, nucleic acid chemistry, immunology laboratory, etc. used herein are all conventional steps widely used in the corresponding fields. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0117] As used herein, the term "isolated" means obtained by artificial means from its natural state. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located has changed or the substance has been separated from the natural environment, or both situations have occurred. For example, for a certain unisolated polynucleotide or polypeptide naturally present in a living animal body, the highly purified same polynucleotide or polypeptide separated from this natural state is called "isolated". The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0118] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic material elements it carries to 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); bacteriophages 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0119] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0120] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 of 6 total positions match). Typically, comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be accomplished by using, e.g., the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). Also, the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0), can be used with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, 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 www.gcg.com) can be used with a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences.

[0121] The writing of the twenty conventional amino acids involved in this article follows conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. 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 the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0122] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well-known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, ionic strength enhancers, osmotic pressure maintaining reagents, absorption delaying reagents, diluents, adjuvants, preservatives, stabilizers, etc. For example, pH regulators include but are not limited to phosphate buffer. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Osmotic pressure maintaining reagents include but are not limited to sugars, NaCl and its analogues. Absorption delaying reagents include but are not limited to monostearate and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Adjuvants include but are not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as complete Freund's adjuvant), etc. Preservatives include but are not limited to various antibacterial and antifungal reagents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and can stabilize the desired activity of the active ingredient in the drug (such as the inhibitory activity against PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, saccharides (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0123] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer deteriorating) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or not. In addition, "treatment" may also refer to prolonging the survival period compared to the expected survival period (if not treated).

[0124] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human.

[0125] Advantages of the invention

[0126] The delivery system of the present invention can significantly improve the release of cargo molecules from endocytic vesicles, thereby significantly enhancing the cytoplasmic delivery efficiency of cargo molecules and enabling cargo molecules to fully exert their corresponding biological functions. 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, treatment, and diagnosis, having broad application prospects and clinical value.

[0127] The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and not to limit the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become apparent to those skilled in the art. Brief Description of the Drawings

[0128] Figure 1 Shows a schematic diagram of the principle for detecting the endocytic vesicle escape efficiency using the Split-GFP system in Example 1.

[0129] Figure 2 Shows a schematic diagram of the structure of the delivery system-GFPβ1-10 protein complex in Example 1.

[0130] Figures 3-4 Shows the SDS-PAGE results of the delivery system-GFPβ1-10 complex in Example 1.

[0131] Figure 5 Shows the flow cytometry analysis results of the delivery system transducing GFPβ1-10 in Example 1.

[0132] Figure 6 Shows the fluorescence microscopy observation results of the delivery system transducing GFPβ1-10 in Example 1.

[0133] Figure 7 Shows the flow cytometry analysis results of the delivery system containing a cleavage site mutation transducing GFPβ1-10 in Example 1.

[0134] Figure 8 Shows the Western blot detection results of the relative proportions of cleaved and uncleaved / intact GFPβ1-10 in cells after transduction with the delivery system in Example 1 and their residence times in cells.

[0135] Figure 9 Shows the Western blot detection results of the relative proportions of cleaved and uncleaved / intact GFPβ1-10 in cells after transduction with the delivery system containing a cleavage site mutation in Example 1.

[0136] Figure 10Shows the flow cytometry analysis results after transduction of the delivery system containing a cleavage site but no pH-sensitive peptide in Example 1.

[0137] Figure 11 Shows the Western blot detection results of the relative proportions of cleaved and uncleaved / intact GFPβ1-10 in cells after transduction of the delivery system containing a cleavage site but no pH-sensitive peptide in Example 1.

[0138] Figure 12 Shows the schematic structural diagram of the delivery system-ZFP9 complex in Example 2.

[0139] Figure 13 Shows the SDS-PAGE results of the delivery system-ZFP9 complex in Example 2.

[0140] Figure 14 Shows the map of the eukaryotic expression plasmid containing the ZFP9 binding site in Example 2.

[0141] Figures 15-16 Shows the flow cytometry analysis results of transduction of ZFP9 by the delivery system in Example 2.

[0142] Figure 17 Shows the schematic structural diagram of the delivery system-Ppm1b complex in Example 3.

[0143] Figure 18 Shows the SDS-PAGE results of the delivery system-Ppm1b complex in Example 3.

[0144] Figure 19 Shows the flow cytometry analysis results of the effect of the delivery system-Ppm1b complex on the ratio of TNF-α-induced cell necrosis in Example 3.

[0145] Figure 20 Shows the schematic structural diagram of the delivery system-Cas9 complex in Example 4.

[0146] Figure 21 Shows the SDS-PAGE results of the delivery system-Cas9 complex in Example 4.

[0147] Figure 22 Shows the schematic diagram of the principle for detecting CRISPR / Cas9 editing efficiency using HEK293T-RFP reporter cells in Example 4.

[0148] Figure 23 Shows the map of the RFP repoter lentiviral plasmid in Example 4.

[0149] Figure 24Shows the flow cytometry analysis results of the gene editing efficiency of the delivery system-Cas9 complex in Example 4.

[0150] Figure 25 Shows the principle of the delivery system based on the adapter method in Example 5.

[0151] Figure 26 Shows the recombinant protein-related clone design of the delivery system based on the adapter method in Example 5.

[0152] Figure 27 Shows the recombinant protein purification results of the delivery system based on the adapter method in Example 5.

[0153] Figure 28 Shows the evaluation of the delivery effect of the delivery system based on the adapter method using the Split-GFP vesicle escape system in Example 5.

[0154] Sequence information

[0155] Information on some of the sequences involved in the present invention is provided in Table 1 below.

[0156] Table 1: Description of sequences

[0157]

[0158] Detailed implementation manners

[0159] The present invention will now be described with reference to the following examples, which are intended to illustrate (but not limit) the present invention.

[0160] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention are basically carried out according 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., Current Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases follows the conditions recommended by the product manufacturers. Those skilled in the art will appreciate that the examples describe the present invention by way of illustration and are not intended to limit the scope of the present invention claimed.

[0161] The sources of the main reagents involved in the following examples are as follows:

[0162] The materials required for clone construction-related reagents are as follows: DNA polymerase (TaKaRa, R040A), DNA recovery kit (TianGen, DP214-03), plasmid mini-prep kit (TianGen, DP103-03), plasmid maxi-prep kit (QIAGEN, 12663), Gibson Assembly Master Mix 5 tubes (NEB, E2611L), DNA marker (ThmeroFisher, SM0331), agarose (Biowest, BW-R0100).

[0163] The materials required for large-scale protein expression are as follows: peptone (BiSIGMA-ALDRICH, T7293-1KG), yeast extract (OXOID, LP0021B), sodium chloride (Xilong Chemical, 10011012AR), IPTG (Inalco, 1758-1400).

[0164] The media required for protein purification are as follows: SP SEPHAROSE FAST FLOW (GE Healthcare, 17-0729-01), NI SEPHAROSE (GE Healthcare, 17-5268-02).

[0165] The reagents required for protein purification and preservation are as follows: glycerol / glycerin / C3H8O3 (SIGMA-ALDRICH, G5516), KCl (Xilong Chemical, 1002007), Na2HPO4·12H2O (Xilong Chemical, 1001067AR), KH2PO4 (Xilong Chemical, 1002048AR500), imidazole (SIGMA-ALDRICH, V900153), Tris base (Seebio, 183995), glucose (Xilong Chemical, 1064008AR500), BCA Protein Assay Kit (Thermo Scientific, 23227).

[0166] The reagents required for cell culture: FBS (GIBCO, 10099-133), DMEM (GIBCO, 11965092), trypsin (AMRESCO, 0458).

[0167] Reagents required for lentivirus packaging and infection: Lentivirus 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);

[0168] Plasmids related to GFPβ1-10, ZFP9, Ppm1b, dsRed, mCherry, and Histone-H3 used in the experiment were all synthesized by the company (Sangon Biotech), and the plasmid pCasKP-hph (Addgene, 117232) for amplifying the Cas9 sequence;

[0169] Other reagents: TNF-α (Novoprotein, CF09), PI (ThmeroFisher, P3566)

[0170] Cell lines: HEK-293T (human renal epithelial cells) and L929 (mouse fibroblasts) were both purchased from ATCC.

[0171] Example 1: Evaluation of the endocytic vesicle escape efficiency of the delivery system based on the Split-GFP system

[0172] In the Split-GFP system, the 11 β-strands of GFP are split into a large fragment (β1-10) and a small fragment (β11), both of which lose their fluorescence activity, but can spontaneously associate and restore the fluorescence performance of GFP if they meet. Based on this, we constructed HEK293T cells stably expressing Histone-β11, and at the same time, GFPβ1-10 with a nuclear localization signal (NLS) was fused and expressed with the endocytic delivery system to be evaluated, and then this stable cell line was transduced. When the delivery system transduces GFPβ1-10, only after successfully escaping from the endocytic vesicle and entering the cytoplasm or nucleus can it bind to GFPβ11 to generate complete GFP. Therefore, the endocytic vesicle escape efficiency can be evaluated by the proportion and relative fluorescence intensity of GFP ( Figure 1 ).

[0173] 1.1 Construction of the expression vector of the delivery system-GFPβ1-10 protein complex

[0174] Construct a recombinant protein expression vector containing TAT (SEQ ID NO: 10), INF7 (SEQ ID NO: 8), a protease cleavage site (Table 2), and a cargo molecule GFPβ1-10 (SEQ ID NO: 23) carrying a nuclear localization signal (NLS). The schematic diagrams of the structures of the recombinant proteins are as shown in Figure 2 shown. The amino acid sequences of each component from the C-terminus to the N-terminus are shown in Table 3 below. The construction method is as follows: First, PCR amplification is used to obtain the nucleic acid sequences encoding TAT, INF7, N, Na, Nb, Nc, Nd, Ne, Nf, mutant N, mutant Ne, and the cargo molecule GFPβ1-10 in the delivery system. The various parts are ligated through multiple rounds of PCR, and in the last round of PCR, an NdeI cleavage site and its overlapping sequence upstream of the NdeI cleavage site corresponding to pET21b(+) are introduced at the 5' end of the fragment through the upstream primer, and a BamHI cleavage site and its overlapping sequence downstream of the BamHI cleavage site corresponding to pET-21b(+) are introduced at the 3' end of the fragment through the downstream primer. The pET-21b(+) plasmid is treated with double digestion by NdeI and BamHI. The insert fragment with the overlapping sequence is ligated to the digested vector pET-21b(+) through GIBSON assembly.

[0175] Table 2: Protease recognition sequences

[0176]

[0177] Table 3: Components contained in the delivery system-cargo molecule complex

[0178]

[0179] 1.2 Expression and purification of the delivery system-GFPβ1-10 complex:

[0180] Transform the expression plasmid described in 1.1 into the expression strain E. coli BL21(DE3); pick a single colony from the transformed plate and inoculate it into 5 ml of LB liquid medium containing ampicillin resistance and culture overnight. Then, take 1 ml of the overnight culture and transfer it to 500 ml of LB liquid medium containing ampicillin resistance, and culture at 37 °C and 180 rpm until the OD of the bacterial liquid 600At around 0.6, then add the inducer IPTG to a final concentration of 0.2 mM, and induce for 8 h at 25 °C; after the induction expression is completed, centrifuge at 4 °C and 7000 g for 10 min to collect the bacterial cells; then resuspend the bacterial cells with 10 ml of protein purification equilibrium buffer (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4 dissolved in 1 L of double-distilled water), and perform ultrasonic disruption. Then centrifuge and take the supernatant, and load it onto the polyhistidine protein purification column of the protein purification system; then use the protein purification elution buffer (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 17 g of imidazole dissolved in 1 L of double-distilled water) to elute the target protein using the protein purification system. The protein concentration can be measured according to a spectrophotometer or a BCA protein concentration assay kit. Each purified fusion protein is aliquoted and stored at -20 °C. The SDS-PAGE results of each protein are as Figures 3-4 shown.

[0181] 1.3 Construction of HEK293T-GFPβ11 cell line

[0182] 1.3.1 Construction of lentiviral plasmid of GFPβ11 cell line:

[0183] The coding sequence of Histone-H3 (SEQ ID NO:25) was obtained by PCR amplification. The coding sequence of GFPβ11 (SEQ ID NO:26) was shorter and was directly designed in the upstream primer. Each component was ligated by multiple rounds of PCR, and in the last round of PCR, a Hind III restriction site and its overlapping sequence upstream of the Hind III restriction site corresponding to the Lenti vector were introduced at the 5' end of the fragment through the upstream primer, and a BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to the Lenti vector were introduced at the 3' end of the fragment through the downstream primer. The Lenti plasmid was digested with Hind III and BamH I. The insert fragment with overlapping sequences was ligated to the digested Lenti vector by GIBSON assembly.

[0184] 1.3.2 Lentivirus packaging, infection and resistant screening of cell line:

[0185] Inoculate HEK-293T cells into a 6-well plate and culture overnight to ensure that the number of cells in each well is about 2*10 7Around / ml; before transfection, the cells were changed to serum-free DMEM medium; 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 5:3:2:1) were added to 300 μl of serum-free DMEM respectively. After gently blowing and mixing evenly, 9 μl of (1:3) X-tremeGENE transfection reagent was added, and after gently blowing and mixing evenly, it was left standing at room temperature for 15 min, then dropped into the cell supernatant. After 8 h, it was changed to DMEM containing 10% FBS for continued culture; after 60 h, the culture supernatant was collected for infection.

[0186] HEK-293T cells were seeded into 12-well plates and cultured overnight. Before lentivirus infection, the number of cells per well was ensured to be approximately 2×10 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 after centrifugation at 2500 rpm for 30 min under sterile conditions in the cell plate, the culture was continued.

[0187] 48 h after lentivirus infection, the cells were passaged at a ratio of 1 / 3, and at the same time, puromycin was added at a concentration of 2.5 μg / ml for resistance screening; the positive cells obtained by screening were cloned to obtain a monoclonal cell line of HEK-293T-Hitone-GFPβ11.

[0188] 1.4 Detection of the endocytic vesicle escape efficiency of the delivery system-GFPβ1-10 complex by the Split-GFP system

[0189] The HEK-293T-Hitone-GFPβ11 cell line obtained in 1.3 was seeded into 12-well plates and cultured overnight. Before protein treatment, the number of cells per well was ensured to be approximately 5×10 6 / ml; the cells were rinsed three times with serum-free DMEM medium, and 100 μl / 5 μM of the delivery system-GFPβ1-10 complex obtained in 1.2 was added in an environment of serum-free medium and incubated for 3 h. After washing three times with heparin solution to remove the proteins adsorbed on the cell surface and not yet endocytosed into the cells, it was changed to DMEM containing 10% FBS for continued culture, and the expression of green fluorescent protein was observed by fluorescence microscopy and analyzed by flow cytometry at 12 h.

[0190] The results of flow cytometry analysis are as Figure 5As shown, the results show that the average fluorescence intensity of cells increases after adding the pH-sensitive peptide INF7 to TAT, demonstrating the membrane-breaking effect of the pH-sensitive peptide on the endocytic vesicle membrane; in particular, after adding the cleavage sites of CTSL or Furin to TAT-INF7, the fluorescence intensity of the cells further increases, and the cleavage site N of CTSL and the cleavage site Ne of Furin have the most significant effects. Fluorescence microscopy observations are as Figure 6 shown. The results show that combining the above two cleavage sites (TINNe-GFPβ1-10) can further significantly improve the endocytic vesicle escape efficiency.

[0191] Mutations were introduced at the key sites in the N or Ne cleavage site to obtain a delivery system-cargo molecule complex containing the mutation. Among them, the sequence of the mutated N is shown in SEQ ID NO:21, and the sequence of the mutated Ne is shown in SEQ ID NO:22; and the transfection efficiency of the mutated delivery system-cargo molecule complex (Mut) and the delivery system-cargo molecule complex without mutation (WT) was compared. The results are as Figure 7 shown. When the key amino acids in the cleavage site were mutated, whether it was a single cleavage site or the combination of double cleavage sites, they all lost their effect of enhancing the vesicle escape efficiency. The above results indicate that the CTSL and Furin cleavage sites can indeed significantly improve the escape efficiency of Cargo in vesicles.

[0192] After washing the cells three times with heparin solution to remove the proteins adsorbed on the cell surface and not yet endocytosed into the cells, the cells at different time points after the start of transduction were collected, the cells were lysed to extract proteins and subjected to SDS-PAGE electrophoresis, and then western blot detection was performed using a monoclonal antibody (Abcam, ab32146) that recognizes GFPβ1-10 to analyze the cleavage and retention of intracellular proteins. The results are as Figure 8As shown, at each time point before 6 h, the total amount of intracellular proteins detected was comparable among the groups of cells, indicating that the pH-sensitive peptide and the cleavage site did not increase the intracellular endocytosis efficiency of the Cargo; cleavage of the proteins containing the cleavage site (TIN-, TINe-, TINNe-) occurred at 30 min, and the amount of cleaved protein did not increase after 3 h. At this time, about 40% of the single-cleavage-site protein was cleaved, while about 70% of the double-cleavage-site protein was cleaved; thereafter, the proteins without a cleavage site (T-, TI-) and the intact proteins with uncleaved cleavage sites began to degrade rapidly due to being directed to lysosomes, and the corresponding bands almost disappeared at 12 h, while the proteins separated from TAT-INF7 after cleavage still remained, and the TINNe group had the most remaining. The above results were consistent with the results of flow cytometry detection. The higher the cleavage efficiency, the more GFPβ1-10-NLS remained undegraded in the cells, the more intact GFP formed by its entry into the nucleus and association with GFPβ11, and the higher the green fluorescence intensity.

[0193] Furthermore, western blot detection was performed on the cleavage analysis of the delivery system-cargo molecule complex containing mutations in the N or Ne cleavage site after transducing cells for 3 h. The results were as Figure 9 shown. The mutated delivery system-cargo molecule complexes (TINm-, TINem- and TINNem-) could not achieve cleavage in the cells. Therefore, combined with Figure 7 the flow cytometry results shown, the inability to achieve cleavage and subsequent escape was the reason for the loss of high-efficiency delivery of the mutated delivery system-cargo molecule complex, and it further confirmed the key role of the CTSL and Furin cleavage sites in the delivery system.

[0194] In addition, flow cytometry detection was also performed on the delivery efficiency of the delivery system complex (TNNe-GFPβ1-10-NLS) lacking the pH-sensitive peptide component. The results were as Figure 10 shown. We found that the average fluorescence intensity after 12 h of transduction was basically the same as that of T-GFPβ1-10-NLS, indicating that even if there was a cleavage site, the lack of a pH-sensitive peptide could not achieve efficient delivery. At the same time, western blot detection found that the presence or absence of a pH-sensitive peptide had no effect on the cleavage efficiency of the delivery system complex in the cells ( Figure 11 ). Combining the above results, we confirmed that only the simultaneous presence of a pH-sensitive peptide and a specific cleavage site in the delivery system could achieve the final high-efficiency delivery.

[0195] Example 2: Application of the delivery system in transducing zinc finger protein ZFP

[0196] 2.1 Construction of the expression vector of the delivery system-zinc finger protein ZFP9 complex

[0197] Construct a recombinant protein expression vector containing TAT, INF7, protease cleavage site, and cargo molecule ZFP9 (SEQ ID NO: 27) carrying a nuclear localization signal (NLS). The structural schematic diagrams of each recombinant protein are as shown in Figure 12 Figure [not provided in the original]. The amino acid sequences of each component are shown in Table 4 below. The construction method is as follows: First, PCR amplification is used to obtain the nucleic acid sequences encoding TAT, INF7, protease cleavage site, and cargo molecule ZFP9 in the delivery system. Then, each part is ligated through multiple rounds of PCR. During the last round of PCR, an NdeI restriction site and its overlapping sequence upstream of the corresponding NdeI restriction site in pET-21b(+) are introduced at the 5' end of the fragment through the upstream primer, and a BamHI restriction site and its overlapping sequence downstream of the corresponding BamHI restriction site in pET-21b(+) are introduced at the 3' end of the fragment through the downstream primer. The pET-21b(+) plasmid is treated with double digestion by NdeI and BamHI. The insert fragment with overlapping sequences is ligated to the digested vector pET-21b(+) through GIBSON assembly.

[0198] Table 4: Components contained in the delivery system-cargo molecule complex

[0199]

[0200] 2.2 Expression and purification of the delivery system-ZFP9 complex

[0201] Transform the expression plasmid described in 2.1 into the expression strain E. coli BL21(DE3); pick a single colony from the transformed plate and inoculate it into 5 ml of LB liquid medium containing ampicillin resistance and culture overnight. Then, take 1 ml of the overnight culture broth and transfer it to 500 ml of LB liquid medium containing ampicillin resistance, and culture at 37°C and 180 rpm until the OD of the broth 600At around 0.6, IPTG was then added to a final concentration of 0.2 mM, and induction was carried out at 25 °C for 8 hours. After the induction of expression was completed, the cells were collected by centrifugation at 4 °C and 7000 g for 10 min. Part of the cells was taken to detect the protein induction expression. Then, the cells were resuspended with 10 ml of protein purification equilibration buffer (50 ml of glycerol, 3.6342 g of Tris(Hydroxymethyl)aminomethane dissolved in 1 L of double-distilled water, pH adjusted to 8.0), and ultrasonic disruption was performed. Then, the supernatant was obtained by centrifugation and loaded onto a Sulphopropyl (SP) cation exchange column of the AKTA protein purification system. Then, gradient elution was carried out using different ratios between the equilibration buffer and the high-salt elution buffer (50 ml of glycerol, 116.88 g of NaCl, 3.6342 g of Tris(Hydroxymethyl)aminomethane dissolved in 1 L of double-distilled water, pH adjusted to 8.0) to obtain the target protein. The protein concentration can be measured using a spectrophotometer or a BCA protein concentration assay kit. Each purified fusion protein was aliquoted and stored at -20 °C. The SDS-PAGE results of each protein are as Figure 13 shown.

[0202] 2.3 Construction of eukaryotic expression plasmid containing ZFP9 binding site

[0203] An expression vector containing the coding sequence of blue fluorescent protein (BFP) and the ZFP9 binding site was constructed, and its structural schematic diagram is as Figure 14 shown. The coding sequence of blue fluorescent protein (SEQ ID NO:29) and the sequence of ZFP9 binding site 6*binding sites (6 binding sites) (SEQ ID NO:30) were obtained by PCR amplification. The two were ligated through two rounds of PCR, and during the second round of PCR, a Hind III restriction enzyme site was introduced at the 5' end of the upstream primer fragment and the overlapping sequence upstream of the corresponding HindIII restriction enzyme site of the pTT5 vector, and a BamHI restriction enzyme site was introduced at the 3' end of the downstream primer fragment and the overlapping sequence downstream of the corresponding BamHI restriction enzyme site of the pTT5 vector. The pTT5 plasmid was digested with Hind III and BamH I. The insert fragment with overlapping sequences was ligated to the digested pTT5 vector through GIBSON assembly to obtain the pTT5-BFP-6BS plasmid.

[0204] 2.4 Detection of the delivery efficiency of zinc finger protein ZFP9 by the delivery system

[0205] HEK293T cells were seeded into 12-well plates and cultured overnight. Before protein treatment, the cell number in each well was ensured to be approximately 5*10 6Around / ml; Co-incubate 100 μL of the delivery system-ZFP9 complex (ZFP9, T-ZFP9, TI-ZFP9, TINNe-ZFP9) obtained in 2.2 and 5 μg of the pTT5-BFP-6BS plasmid obtained in 2.3 at 37 °C for 30 min to fully form the complex, and use X-tremeGENE transfection reagent (Roche) as a positive control (mix 5 μg of the plasmid with 15 μl of the transfection reagent and transfect the cells under serum-free conditions, and then change to 10% FBS DMEM for continued culture after 8 h); Rinse the cells three times with serum-free DMEM medium, then add the complex and incubate for 3 h. Wash three times with heparin solution to remove the proteins adsorbed on the cell surface that have not been endocytosed into the cells, and then change to 10% FBS DMEM medium for continued culture. Change to 10% FBS DMEM for continued culture, and perform flow cytometry analysis of the expression of blue fluorescent protein at 12 h, 24 h, 36 h, and 48 h after changing the medium.

[0206] The results of flow cytometry detection are as Figures 15-16 shown. The plasmid bound to ZFP9 can only complete the transcription process when it enters the cell nucleus. Therefore, only when more ZFP9 escapes from the endocytic vesicles can it carry the bound plasmid into the cell nucleus to start the transcription process and then express blue fluorescent protein. Compared with T-ZFP9, the addition of the pH-sensitive peptide INF7 (TI-ZFP9) can increase the blue fluorescence ratio by about 10% (p = 0.035), but it is still at a relatively low level, that is, most of the complexes fail to escape and enter the nucleus. On this basis, after introducing protease cleavage sites into the system (TINNe-ZFP9), the results show that the ratio of blue fluorescent cells increases significantly, reaching about 65% at 48 h (p = 0.018), and it can be comparable to the Roche transfection reagent X-tremeGENE. Thus, in the endocytic process of the delivery system carrying the ZFP9 / pTT5-BFP-6BS complex into the cells, the addition of the CTSL and Furin specific cleavage sites N and Ne significantly promotes the escape process of the carried ZFP9, and more complexes can enter the nucleus to complete transcription and then express BFP fluorescence.

[0207] Example 3: Application of the delivery system in transducing protein phosphatase Ppm1b

[0208] After TNF-α binds to cell surface receptors, it induces the phosphorylation of RIP3 and forms a necrosome, a multi-protein complex. Phosphorylated RIP3 in the necrosome recruits and phosphorylates Mlkl, after which the cell enters the necrosis program. During this process, intracellular protein phosphatase 1B (Ppm1b) can inhibit necroptosis by dephosphorylating RIP3. Given that necroptosis has been found to be closely related to the occurrence of various diseases such as inflammatory diseases, ischemia-reperfusion injury, and neurodegenerative diseases, Ppm1b protein shows great potential in the treatment of the above diseases related to necroptosis.

[0209] 3.1 Delivery system - Construction of the Ppm1b protein complex expression vector

[0210] Construct a recombinant protein expression vector containing TAT, INF7, protease cleavage sites, and the cargo molecule Ppm1b (SEQ ID NO: 31). The schematic diagrams of the structures of each recombinant protein are as Figure 17 shown, and the amino acid sequences of each component are shown in Table 6 below. The construction method is as follows: Obtain the nucleic acid sequences of TAT, INF7, protease cleavage sites, and Ppm1b by PCR amplification, and ligate each part through multiple rounds of PCR. During the last round of PCR, introduce an NdeI restriction site at the 5' end of the fragment and the overlapping sequence upstream of the corresponding NdeI restriction site of pET-21b(+), and introduce a BamHI restriction site at the 3' end of the fragment and the overlapping sequence downstream of the corresponding BamHI restriction site of pET-21b(+) through the downstream primer. The pET-21b(+) plasmid is treated with double digestion by NdeI and BamHI. Connect the insert fragment with overlapping sequences to the digested vector pET-21b(+) by GIBSON assembly.

[0211] Table 5: Components included in the delivery system - cargo molecule complex

[0212]

[0213] 3.2 Delivery system - Expression and purification of the Ppm1b complex

[0214] Transform the expression plasmid described in 3.1 into the expression strain E. coli BL21(DE3); pick a single colony from the transformed plate and inoculate it into 5 ml of LB liquid medium containing ampicillin resistance and culture overnight. Then, take 1 ml of the overnight culture broth and transfer it to 500 ml of LB liquid medium containing ampicillin resistance, and culture at 37°C and 180 rpm until the OD of the broth 600At around 0.6, then add IPTG to a final concentration of 0.2 mM and induce at 25 °C for 8 h. After the induction of expression, centrifuge the cells at 4 °C and 7000 g for 10 minutes to collect the cells. Take a part of the cells to detect the protein induction expression. Then resuspend the cells with 10 ml of protein purification equilibrium buffer (50 ml of C3H8O3, 3.6342 g of Tris(Hydroxymethyl)aminomethane dissolved in 1 L of double-distilled water, adjust the pH to 8.0), and perform ultrasonic disruption. Then centrifuge and take the supernatant, and load it onto a Sulphopropyl (SP) cation exchange column of the AKTA protein purification system. Then use a gradient elution with different ratios between the equilibrium buffer and the high-salt elution buffer (50 ml of C3H8O3, 116.88 g of NaCl, 3.6342 g of Tris(Hydroxymethyl)aminomethane dissolved in 1 L of double-distilled water, adjust the pH to 8.0) to obtain the target protein. The protein concentration can be measured according to a spectrophotometer or a BCA protein concentration assay kit. Each purified fusion protein is aliquoted and stored at -20 °C. The SDS-PAGE results of each protein are as Figure 18 shown.

[0215] 3.3 Effect of the Delivery System-Ppm1b Complex on the Ratio of TNF-α-induced Cell Necrosis

[0216] Inoculate L929 cells into a 12-well cell culture plate and culture overnight. Ensure that the number of cells per well is about 2×10 6 / ml before protein treatment. Rinse the cells three times with serum-free DMEM medium, and then add 100 μl / 5 μM of the delivery system proteins (Ppm1b, T-Ppm1b, TI-Ppm1b, TINNe-Ppm1b) obtained in 3.2 to the serum-free medium. After incubation for 3 h, add 1 ml of 20 ng / ml TNF-α and 20 mM z-VAD diluted with 10% FBS DMEM, incubate for 10 h, collect the cells for PI staining, and analyze the cells by flow cytometry to observe the ratio of cell necrosis. Use lentiviral transduction of Ppm1b (Lenti-Ppm1b) and lentivirus (Lenti-vec) as controls. Package the lentivirus carrying the Ppm1b expression sequence and the control lentivirus collected on HEK-293T cells. After infecting L929 cells for 24 h, to allow sufficient intracellular expression of Ppm1b, replate the infected L929 cells and wait for TNF-α stimulation.

[0217] The results are as Figure 19As shown, compared with simple T-Ppm1b, the addition of the pH-sensitive peptide INF7 (TI-Ppm1b) only slightly inhibits cell necrosis, but still remains at a relatively low level. On this basis, after introducing protease cleavage sites into the system (TINNe-Ppm1b), the ability of the TINNe group to inhibit cell necrosis can reach the level of the lentivirus group, and the cell necrosis rate can be greatly reduced to about 25%. Therefore, during the endocytosis process of the delivery system carrying the Ppm1b complex into cells, the addition of the CTSL and Furin specific sites N and Ne significantly promotes the escape process of the carried Ppm1b. More Ppm1b enters the cytoplasm, inhibits the phosphorylation process of RIP3, and reduces the cell necrosis ratio.

[0218] Example 4: Application of the Delivery System in the Gene Editing Engineering Enzyme Cas9

[0219] The CRISPR / Cas9 gene editing system binds the Cas9 protein through sgRNA, specifically recognizes the target site through sgRNA, Cas9 binds and cleaves the DNA double-stranded molecule, and realizes the editing of the target gene through non-homologous end joining or homologous end repair. In this system, Cas9 must enter the nucleus to complete its function. Based on this, we fused and expressed the Cas9 protein with the delivery system to achieve gene editing on eukaryotic cells.

[0220] 4.1 Construction of the Expression Vector of the Delivery System-Cas9 Protein Complex

[0221] Construct a recombinant protein expression vector containing TAT, INF7, protease cleavage sites, and the cargo molecule Cas9 (SEQ ID NO: 33) carrying a nuclear localization signal (NLS). The schematic diagrams of the structures of each recombinant protein are as Figure 20 shown, and the amino acid sequences of each component are shown in Table 6 below. The construction method is as follows: The nucleic acid sequences encoding TAT, INF7, protease cleavage sites N and Ne, and Cas9 are obtained by PCR amplification, and each part is ligated through multiple rounds of PCR. During the last round of PCR, an NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) are introduced at the 5' end of the fragment through the upstream primer, and a BamHI restriction site and its overlapping sequence (overlap) downstream of the BamHI restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment through the downstream primer. The pET-21b(+) plasmid is treated with double digestion by NdeI and BamHI. The insertion fragment with overlapping sequences is ligated to the digested vector pET-21b(+) through GIBSON assembly.

[0222] Table 6: Components Contained in the Delivery System-Cargo Molecule Complex

[0223]

[0224] 4.2 Delivery System - Expression and Purification of Cas9 Complex

[0225] Initial purification by nickel column: Transform the expression plasmid described in 4.1 into the expression strain E. coli BL21(DE3); Pick a single colony from the transformed plate and inoculate it into 5 ml of LB liquid medium containing ampicillin resistance and culture overnight. Then, take 1 ml of the overnight culture and transfer it to 500 ml of LB liquid medium containing ampicillin resistance, and culture at 37 °C and 180 rpm until the OD of the bacterial solution 600 is about 0.6. Then add the inducer IPTG to a final concentration of 0.2 mM and induce at 25 °C for 8 h; After the induction expression is completed, centrifuge at 4 °C and 7000 g for 10 minutes to collect the bacterial cells; Then resuspend the bacterial cells with 10 ml of protein purification equilibration buffer (5% glycerol, 30 mM Tris-HCl pH 8.0, 50 mM glycerol, 500 mM sodium chloride, 25 mM glucose) and perform ultrasonic disruption. Then centrifuge to take the supernatant and load it onto the polyhistidine protein purification column of the protein purification system; Then use the protein purification system to elute the target protein with protein purification elution buffer (5% glycerol, 30 mM Tris-HCl pH 8.0, 50 mM glycerol, 500 mM sodium chloride, 25 mM glucose, 250 mM imidazole).

[0226] Purification by cation exchange column: Dialyze the target protein collected by initial purification with nickel column into the equilibration buffer (30 mM Tris-HCl pH 8.0, 50 mM glycerol, 250 mM sodium chloride, 25 mM glucose, adjust the pH to 7.2), and load it onto the Sulphopropyl (SP) cation exchange column of the AKTA protein purification system; Then perform gradient elution to obtain the target protein according to different ratios between the equilibration buffer and the high-salt elution buffer (30 mM Tris-HCl pH 8.0, 50 mM glycerol, 2 M sodium chloride, 25 mM glucose, adjust the pH to 7.2). The protein concentration can be measured according to the spectrophotometer or the BCA protein concentration assay kit. After aliquoting each purified fusion protein, store it at -20 °C. The SDS-PAGE results of each protein are as Figure 21 shown.

[0227] 4.3 Construction of HEK293T-RFP reporter cell line

[0228] After Cas9 binds to sgRNA, it can specifically recognize the target site. In the absence of a donor, non-homologous end joining occurs. Therefore, by means of lentiviral infection, the sgRNA recognition site and two red fluorescent protein genes that are not in the reading frame (there is a difference of one G between dsRed and mCherry) are integrated into the genome of HEK-293T cells. If Cas9 causes homologous end joining of the DNA sequence at the sgRNA recognition site, it will cause the red fluorescent protein genes that were originally not in the reading frame to enter the reading frame and be expressed, making the cells change from non-fluorescent to red fluorescent. The efficiency of the delivery system for transducing the gene engineering enzyme Cas9 can be evaluated by whether red fluorescence is produced and the number of red fluorescent cells ( Figure 22 ).

[0229] 4.3.1 Construction of lentiviral plasmid for RFP repoter cell line

[0230] The coding sequences of dsRed (SEQ ID NO:35) and mCherry (SEQ ID NO:36) were obtained by PCR amplification. The DNA sequence of the sgRNA recognition site (SEQ ID NO:37) is relatively short and was directly designed in the primers. Each component was ligated through multiple rounds of PCR, and during the last round of PCR, a Hind III restriction site and its overlapping sequence upstream of the Hind III restriction site corresponding to the Lenti vector were introduced at the 5' end of the upstream fragment through the upstream primer, and a BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to the Lenti vector were introduced at the 3' end of the downstream fragment through the downstream primer. The Lenti plasmid was digested with HindIII and BamH I. The inserted fragment with overlapping sequences was ligated to the digested Lenti vector through GIBSON assembly, and the plasmid map of the successfully constructed plasmid ( Figure 23 ).

[0231] 4.3.2 Lentivirus packaging, infection and cell line resistance screening

[0232] HEK293T cells were seeded into 6-well plates and cultured overnight. Before plasmid transfection, ensure that the number of cells in each well is approximately 2*10 7Around / ml; before transfection, the cells were replaced with serum-free DMEM medium; in 300 μl of serum-free DMEM, 1.5 μg of Lenti recombinant plasmid (RFP reporter), 0.75 μg of pMDL plasmid, 0.45 μg of pVSV-G plasmid, and 0.3 μg of pREV plasmid (mass ratio 5:3:2:1) were added respectively, gently blown and mixed evenly, left standing for 5 min, then 9 μl of X-tremeGENE transfection reagent was added, gently blown and mixed evenly, left standing at room temperature for 15 min, and then added to the cell supernatant. After 8 h, it was replaced with DMEM containing 10% FBS for continuous culture; after 60 h, the culture supernatant was collected and stored at 4 °C.

[0233] HEK293T cells were seeded into 12-well plates and cultured overnight. Before lentivirus infection, ensure that the number of cells in each well is approximately 2×10 6 / ml (50% density); discard the cell culture supernatant, add 300 μl of lentivirus (moi = 3) and 700 μl of 10% FBS DMEM, add polybrene at a concentration of 10 μg / ml, and continue to culture after centrifugation at 2500 rpm for 30 min under no conditions.

[0234] 48 h after lentivirus infection, the cells were passaged at a ratio of 1 / 3, and at the same time, puromycin was added at a concentration of 2.5 μg / ml for resistance screening; the positive cells obtained by screening were cloned to obtain a monoclonal cell line of HEK293T-RFP reporter.

[0235] 4.3.3 Construction of GM3-gRNA transcription plasmid

[0236] The introduction of gRNA uses the method of transcribing into gRNA in cells after transfection with transcription plasmid. The DNA sequence corresponding to gRNA is generated by primer annealing and mutual connection. The sticky ends of AflII restriction enzyme sites are directly introduced during the primer design process; the gRNA-cloning vector is treated with AflII single enzyme digestion; the vector and the inserted fragment are ligated by T4 DNA ligase using their respective sticky ends.

[0237] 4.4 Evaluation of the editing efficiency of the delivery system - Cas9 complex

[0238] Transfection of gRNA transcription plasmid: The HEK-293T-RFP reporter cell line was seeded into 12-well plates and cultured overnight. Before transfection, ensure that the number of cells in each well is approximately 2.5×10 6Around / ml; Replace the cells with serum-free DMEM before transfection; Add 1 μg of the gRNA-GM3 transcription plasmid to 100 μl of serum-free DMEM, gently pipette to mix evenly, let stand for 5 min, add 3 μl of X-tremeGENE transfection reagent, gently pipette to mix evenly and then let stand for 15 min, add it to the cell supernatant, and replace it with 10% FBS DMEM after 8 h.

[0239] Transduction of Cas9 by the delivery system: 12 h after transfection of the gRNA transcription plasmid (4 h after changing to serum-containing DMEM), rinse the cells three times with serum-free DMEM, and add the delivery system-Cas9 complex obtained in 4.2 at 5 μM in the serum-free DMEM environment and incubate for 3 h; Replace it with 10% FBS DMEM and continue culturing, and observe and perform flow cytometry analysis of the expression of red fluorescent protein at 48 h.

[0240] The flow cytometry results are as Figure 24 shown. After transduction with T-Cas9, the proportion of red fluorescent cells is relatively low (3.7%), that is, most of the Cas9 protein fails to escape and enter the nucleus; the addition of the pH-sensitive peptide INF7 (TI-Cas9) can increase the proportion of red fluorescent cells to 9%; on this basis, after introducing protease cleavage sites into the system (TINNE-Cas9), the number of cells expressing red fluorescent protein increases significantly and can reach about 14% at 48 h. Therefore, during the endocytosis process of the delivery system carrying Cas9 into the cell, the addition of the CTSL and Furin specific sites N and Ne significantly promotes the escape process of the carried Cas9. More Cas9 enters the nucleus under the action of the nuclear localization signal, binds to the GM3 sgRNA formed by intracellular transcription after binding, specifically recognizes the target sequence, and undergoes homologous end joining near this site, so that the red fluorescent gene enters the reading frame and is expressed.

[0241] Example 5: Establishment of a delivery system based on non-covalent linkage

[0242] In addition to fusion expression, the connection mode between the fusion protein and the cargo of the present invention can also be non-covalently linked through protein domains, which we call the Adapter interaction pair, such as the heterodimeric leucine zipper with strong interaction force ( Figure 25)。Two antiparallel leucine zipper domains can spontaneously bind to form oligomers due to their interlocking spatial characteristics and charge arrangements. According to research reports, NZ and CZ are such a pair of leucine zippers that can bind to each other. We attempted to fuse and express the NZ domain with the cell entry delivery system of the present invention (TINNe-NZ), and fuse and express the CZ domain with GFPβ1-10 carrying a nuclear localization signal (NLS) as Cargo (CZ-GFPβ1-10-NLS). After mixing and incubating the above two proteins to bind to each other, HEK293T cells stably expressing Histone-β11 were transduced, and the endocytic vesicle escape efficiency could be evaluated by the proportion and relative fluorescence intensity of GFP. Thus, the feasibility of the NZ-CZ pair of adapters to connect the fusion protein and the cargo was evaluated.

[0243] 5.1 Construction of expression vectors for the delivery system-NZ fusion protein and CZ-GFPβ1-10 fusion protein

[0244] Based on the pET32a vector, an expression vector for a recombinant protein containing TAT, INF7, a protease cleavage site, and the NZ domain was constructed, and an expression vector for a recombinant protein carrying the cargo molecule GFPβ1-10 with the CZ domain and a nuclear localization signal (NLS) was constructed, with TrxA added as a solubility-enhancing tag. The schematic diagram of the recombinant protein is as Figure 26As shown, the amino acid sequences of the delivery system are shown in the following table. The construction method is as follows: First, using the previously described vector as a template, the nucleic acid sequences encoding TAT, INF7, N, Ne, and NZ sequences in the delivery system, as well as the CZ sequence and the cargo molecule sGFPβ1-10, are obtained by PCR amplification. For TrxA-TINNe-NZ, in the last round of PCR, a BamHI restriction site and its overlapping sequence upstream of the BamHI restriction site corresponding to pET-32a(+) are introduced at the 5' end of the fragment through the upstream primer, and a HindIII restriction site and its overlapping sequence downstream of the HindIII restriction site corresponding to pET-32a(+) are introduced at the 3' end of the fragment through the downstream primer. The pET-32a(+) plasmid is digested with BamHI and HindIII. The insert fragment with the overlapping sequence is ligated to the digested vector pET-32a(+) by Gibson assembly; for CZ-GFPβ1-10, in the last round of PCR, an NdeI restriction site and its overlapping sequence upstream of the NdeI restriction site corresponding to pET21b(+) are introduced at the 5' end of the fragment through the upstream primer, and a BamHI restriction site and its overlapping sequence downstream of the BamHI restriction site corresponding to pET-21b(+) are introduced at the 3' end of the fragment through the downstream primer. The pET-21b(+) plasmid is digested with NdeI and BamHI. The insert fragment with the overlapping sequence is ligated to the digested vector pET-21b(+) by Gibson assembly.

[0245] Table 7: Components included in the delivery system

[0246]

[0247] 5.2 Expression and purification of the delivery system-NZ fusion protein and the CZ-GFPβ1-10 fusion protein

[0248] The expression plasmid described in 5.1 is transformed into the expression strain E. coli BL21(DE3); single colonies are picked from the transformed plate and inoculated into 5 ml of LB liquid medium containing ampicillin resistance and cultured overnight. Then, 1 ml of the overnight culture is transferred to 500 ml of LB liquid medium containing ampicillin resistance and cultured at 37°C and 180 rpm until the OD of the bacterial solution 600At around 0.6, then add IPTG inducer to a final concentration of 0.2 mM and induce at 25 °C for 8 h; after the induction expression is completed, centrifuge at 4 °C and 7000 g for 10 min to collect the bacterial cells; then resuspend the bacterial cells with 10 ml of protein purification equilibration buffer (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4 dissolved in 1 L of double-distilled water) and perform ultrasonic disruption. Then centrifuge to take the supernatant and load it onto the polyhistidine protein purification column of the protein purification system; then use the protein purification system to elute the target protein with protein purification elution buffer Elution buffer 2# (50 ml of glycerol, 8 g of NaCl, 0.201 g of KCl, 1.44 g of Na2HPO4, 0.24 g of KH2PO4, 17 g of imidazole dissolved in 1 L of double-distilled water). The protein concentration can be measured according to a spectrophotometer or a BCA protein concentration assay kit. After aliquoting each purified fusion protein, store it at -20 °C. The SDS-PAGE results of each protein are as Figure 27 shown.

[0249] 5.3 Detection of the delivery efficiency of the adapter-based delivery system using the Split-GFP system

[0250] Inoculate the HEK-293T-Hitone-GFPβ11 cell line obtained above into a 12-well plate and culture overnight. Ensure that the number of cells in each well is about 5×10 6 / ml before protein treatment; rinse the cells three times with serum-free DMEM medium. The settings of the control group and the experimental group and the dosage of the protein (obtained in 5.2) are shown in the following table. Among them, before incubating with the cells, the two proteins in the control group 2 and the experimental group are mixed at room temperature in serum-free medium for 10 min. Incubate for 3 h in a serum-free medium environment, wash three times with heparin solution to remove the protein adsorbed on the cell surface and not yet endocytosed into the cells, and then change to DMEM medium containing 10% FBS for continued culture. Perform fluorescence microscopy observation and flow cytometry analysis of the proportion and MFI of green fluorescence-positive cells at 12 h.

[0251] The results of flow cytometry analysis are as Figure 28As shown, the results showed that the MFIs of the two control groups (CZ-GFPβ1-10 / TrxA-TINNe + CZ-GFPβ1-10) were both lower than 5, and the MFI of control group 2 (TrxA-TINNe + CZ-GFPβ1-10) was slightly higher than that of control group 1. We speculated that this was due to the non-specific adsorption of TrxA-TINNe itself to CZ-GFPβ1-10, so that the cargo CZ-GFPβ1-10 was brought into the cell by the action of TINNe and endocytic vesicle escape occurred. The MFI value of the TrxA-TINNe-NZ + CZ-GFPβ1-10 group was close to 20, significantly higher than that of the control group, indicating that under the action of NZ and CZ, TrxA-TINNe bound to GFPβ1-10 to a certain extent, and through the action of TINNe, GFPβ1-10 entered the cell and escaped. Although the fluorescence intensity of the adpater ligation method was still lower than that of the fusion expression ligation method of TINNe-GFPβ1-10, this experiment confirmed that based on NZ-CZ or a similar adapter, it could be used as a ligation method between the delivery system and the cargo, and could effectively deliver the cargo into the cell and escape from the vesicle.

[0252] Table 8: Settings of control groups and experimental groups

[0253]

[0254] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and these changes are all within the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof. SEQUENCE LISTING <110> Xiamen University Xiamen Innovax Biotech Co., Ltd. <120> Complex for intracellular delivery of molecules <130> IDC200146 <150> CN201910624609.9 <151> 2019-07-11 <160> 54 <170> PatentIn version 3.5 <210> 1 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence - 1 <220> <221> MISC_FEATURE <222> (2)..(2) <223> X = any amino acid <220> <221> MISC_FEATURE <222> (3)..(3) <223> X = K or R <400> 1 Arg Xaa Xaa Arg 1 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence - 2 <220> <221> MISC_FEATURE <222> (3)..(3) <223> X = any amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> X = K or R <400> 2 Arg Arg Xaa Xaa Arg 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence - 3 <400> 3 Arg Arg His Lys Arg [[ID=7,4]]1 5 <210> 4 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence - 4 (Ne) <400> 4 Gln Ser Val Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val 1 5 10 15 <210> 5 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Ne <400> 5 cagagcgttg caagcagccg tcgtcataaa cgttttgcag gtgtt 45 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <220> <223> CTSL recognition sequence N <400> 6 Asn Asn Thr His Asp Leu Val Gly Asp Val Arg Leu Ala Gly Val 1 5 10 15 <210> 7 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding N <400> 7 aacaacactc atgaccttgt cggtgatgtg agattagccg gagtt 45 <210> 8 <211> 23 <212> PRT <213> Artificial sequence <220> <223> INF7 <400> 8 Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly 20 <210> 9 <211> 69 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding INF7 <400> 9 ggcctgttcg aagcaataga aggtttcata gaaaatggtt gggagggaat gatagacggt 60 tggtacggt 69 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Tat(48 - 60) <400> 10 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln 1 5 10 <210> 11 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Tat(48 - 60) <400> 11 ggccgtaaga agcggagaca gcgacgaaga ccgccgcag 39 <210> 12 <211> 56 <212> PRT <213> Artificial sequence <220> <223> Fusion protein TIN <400> 12 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Leu Phe 1 5 10 15 Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp 20 25 30 Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn Thr His Asp Leu Val 35 40 45 Gly Asp Val Arg Leu Ala Gly Val 50 55 <210> 13 <211> 56 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein TINe <400> 13 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Leu Phe 1 5 10 15 Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp 20 25 30 Gly Trp Tyr Gly Gly Gly Gly Gly Ser Gln Ser Val Ala Ser Ser Arg 35 40 45 Arg His Lys Arg Phe Ala Gly Val 50 55 <210> 14 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein TINNe <400> 14 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Gly Leu Phe 1 5 10 15 Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp 20 25 30 Gly Trp Tyr Gly Gly Gly Gly Gly Ser Asn Asn Thr His Asp Leu Val 35 40 45 Gly Asp Val Arg Leu Ala Gly Val Gly Gly Gly Gly Ser Gln Ser Val 50 55 60 Ala Ser Ser Arg Arg His Lys Arg Phe Ala Gly Val 65 70 75 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NLS <400> 15 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 16 <211> 21 <212> PRT <213> Artificial sequence <220> <223> CTSL recognition sequence Na <400> 16 Ala Arg Ala Tyr Gly Phe Arg Gly Pro Gly Pro Gln Leu Arg Arg Gly 1 5 10 15 Trp Arg Pro Ser Ser 20 <210> 17 <211> 24 <212> PRT <213> Artificial sequence <220> <223> CTSL recognition sequence Nb <400> 17 Glu Glu Asp Asn Arg Asp Ser Ser Met Lys Leu Ser Phe Arg Ala Arg 1 5 10 15 Ala Tyr Gly Phe Arg Gly Pro Gly 20 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence Nc <400> 18 Gly Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile 1 5 10 15 Arg <210> 19 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Furin recognition sequence Nd <400> 19 Glu Asp Thr Asp Phe Ala Arg Lys Asn Lys Ile Leu Tyr Thr Gln Gln 1 5 10 15 Val His Gln Thr 20 <210> 20 <211> 9 <212> PRT <213> Artificial sequence <220> <223> CTSD recognition sequence Nf <400> 20 Lys Pro Ile Leu Phe Phe Arg Leu Lys 1 5 <210> 21 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Mutated N <400> 21 Asn Asn Thr His Asp Leu Val Gly Asp Val Gly Leu Ala Gly Val 1 5 10 15 <210> 22 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Mutated Ne <400> 22 Gln Ser Val Ala Ser Ser Arg Arg His Lys Gly Phe Ala Gly Val 1 5 10 15 <210> 23 <211> 228 <212> PRT <213> Artificial sequence <220> <223> GFPβ1-10-NLS <400> 23 Met Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Ile Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Ser Phe Lys Asp Asp Gly Lys Tyr Lys Thr Arg Ala Val 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Thr Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Thr Val Arg His Asn Val Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Thr Val Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys His His His His His His Pro Lys Lys 210 215 220 Lys Arg Lys Val 225 <210> 24 <211> 687 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding GFPβ1-10-NLS <400> 24 atgggcagca aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat 60 ggtgatgtta atgggcacaa attttctgtc agaggagagg gtgaaggtga tgctacaatc 120 ggaaaactca cccttaaatt tatttgcact actggaaaac tacctgttcc atggccaaca 180 cttgtcacta ctctgaccta tggtgttcaa tgcttttccc gttatccgga tcacatgaaa 240 aggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg cactatatct 300 ttcaaagatg acgggaaata caagacgcgt gctgtagtca agtttgaagg tgataccctt 360 gttaatcgta tcgagttaaa gggtactgat tttaaagaag atggaaacat tctcggacac 420 aaactcgagt acaactttaa ctcacacaat gtatacatca cggcagacaa acaaaagaat 480 ggaatcaaag ctaacttcac agttcgccac aacgttgaag atggttccgt tcaactagca 540 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 600 tacctgtcga cacaaactgt cctttcgaaa gatcccaacg aaaagcacca ccaccaccac 660 caccccaaga agaagaggaa ggtgtaa 687 <210> 25 <211> 411 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding Histone-H3 <400> 25 atggctcgca ctaagcaaac tgctcggaag tctactggtg gcaaggcgcc acgcaaacag 60 ttggccacta aggcagcccg caaaagcgct ccggccaccg gcggcgtgaa aaagccccac 120 cgctaccggc cgggcaccgt ggctctgcgc gagatccgcc gttatcagaa gtccactgaa 180 ctgcttattc gtaaactacc tttccagcgc ctggtgcgcg agattgcgca ggactttaaa 240 acagacctgc gtttccagag ctccgctgtg atggctctgc aggaggcgtg cgaggcctac 300 ttggtagggc tatttgagga cactaacctg tgcgccatcc acgccaagcg cgtcactatc 360 atgcccaagg acatccagct cgcccgccgc atccgcggag agagggcgtg a 411 <210> 26 <211> 47 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding GFPβ11 <400> 26 cgggaccaca tggtgctgca cgagtacgtg aacgccgccg gcatcac 47 <210> 27 <211> 98 <212> PRT <213> Artificial sequence <220> <223> ZFP9-NLS <400> 27 Val Ser Arg Pro Gly Glu Arg Pro Phe Gln Cys Arg Ile Cys Met Arg 1 5 10 15 Asn Phe Ser Asp Lys Thr Lys Leu Arg Val His Thr Arg Thr His Thr 20 25 30 Gly Glu Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Val 35 40 45 Arg His Asn Leu Thr Arg His Leu Arg Thr His Thr Gly Glu Lys Pro 50 55 60 Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Gln Ser Thr Ser Leu 65 70 75 80 Gln Arg His Leu Lys Thr His Leu Arg Gly Ser Pro Lys Lys Lys Arg 85 90 95 Lys Val <210> 28 <211> 294 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding ZFP9-NLS <400> 28 gtctctagac ccggggagcg ccccttccag tgtcgcattt gcatgcggaa cttttcggat 60 aaaactaaat tgagagttca tacccgtact cataccggtg aaaaaccgtt tcagtgtcgg 120 atctgtatgc gaaatttctc cgttagacat aatttgacta gacatctacg tacgcacacc 180 ggcgagaagc cattccaatg ccgaatatgc atgcgcaact tcagtcaatc tacttctttg 240 caaagacacc taaaaaccca cctgagagga tcccccaaga agaagcgtaa ggtg 294 <210> 29 <211> 714 <212> DNA <213> Artificial sequence <220> <223> Sequence encoding BFP <400> 29 atggtgtcta agggcgaaga gctgattaag gagaacatgc acatgaagct gtacatggag 60 ggcaccgtgg acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag 120 ggcacccaga ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac 180 atcctggcta ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc 240 cccgacttct tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac 300 gaagacgggg gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc 360 tacaacgtca agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa 420 acactcggct gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc 480 agaaacgaca tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa cgccaagacc 540 acatatagat ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac 600 tacagactgg aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg 660 gcagtggcca gatactgcga cctccctagc aaactggggc acaagcttaa ttga 714 <210> 30 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> ZFP9 binding site sequence <400> 30 tgtagatgga g 11 <210> 31 <211> 378 <212> PRT <213> Artificial sequence <220> <223> Ppm1b <400> 31 Met Gly Ala Phe Leu Asp Lys Pro Lys Thr Glu Lys His Asn Ala His 1 5 10 15 Gly Ala Gly Asn Gly Leu Arg Tyr Gly Leu Ser Ser Met Gln Gly Trp 20 25 30 Arg Val Glu Met Glu Asp Ala His Thr Ala Val Val Gly Ile Pro His 35 40 45 Gly Leu Asp Asn Trp Ser Phe Phe Ala Val Tyr Asp Gly His Ala Gly 50 55 60 Ser Arg Val Ala Asn Tyr Cys Ser Thr His Leu Leu Glu His Ile Thr 65 70 75 80 Thr Asn Glu Asp Phe Arg Ala Ala Asp Lys Ser Gly Ser Ala Leu Glu 85 90 95 Pro Ser Val Glu Ser Val Lys Thr Gly Ile Arg Thr Gly Phe Leu Lys 100 105 110 Ile Asp Glu Tyr Met Arg Asn Phe Ser Asp Leu Arg Asn Gly Met Asp 115 120 125 Arg Ser Gly Ser Thr Ala Val Gly Val Met Val Ser Pro Thr His Met 130 135 140 Tyr Phe Ile Asn Cys Gly Asp Ser Arg Ala Val Leu Cys Arg Asn Gly 145 150 155 160 Gln Val Cys Phe Ser Thr Gln Asp His Lys Pro Cys Asn Pro Val Glu 165 170 175 Lys Glu Arg Ile Gln Asn Ala Gly Gly Ser Val Met Ile Gln Arg Val 180 185 190 Asn Gly Ser Leu Ala Val Ser Arg Ala Leu Gly Asp Tyr Asp Tyr Lys 195 200 205 Cys Val Asp Gly Lys Gly Pro Thr Glu Gln Leu Val Ser Pro Glu Pro 210 215 220 Glu Val Tyr Glu Ile Val Arg Ala Glu Glu Asp Glu Phe Val Val Leu 225 230 235 240 Ala Cys Asp Gly Ile Trp Asp Val Met Ser Asn Glu Glu Leu Cys Glu 245 250 255 Phe Val Lys Ser Arg Leu Glu Val Ser Asp Asp Leu Glu Asn Val Cys 260 265 270 Asn Trp Val Val Asp Thr Cys Leu His Lys Gly Ser Arg Asp Asn Met 275 280 285 Ser Val Val Leu Val Cys Phe Ser Asn Ala Pro Lys Val Ser Glu Glu 290 295 300 Ala Val Lys Arg Asp Ser Glu Leu Asp Lys His Leu Glu Ser Arg Val 305 310 315 320 Glu Glu Ile Met Gln Lys Ser Gly Glu Glu Gly Met Pro Asp Leu Ala 325 330 335 His Val Met Arg Ile Leu Ser Ala Glu Asn Ile Pro Asn Leu Pro Pro 340 345 350 Gly Gly Gly Leu Ala Gly Lys Arg His Val Ile Glu Ala Val Tyr Ser 355 360 365 Arg Leu Asn Pro His Lys Asp Asn Asp Gly 370 375 <210> 32 <211> 1134 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Ppm1b <400> 32 atgggtgcat ttttggataa acccaaaact gaaaagcaca atgctcacgg tgctgggaat 60 ggtctgcgtt atggcctgag cagtatgcaa ggatggagag tagaaatgga agatgcacac 120 acagctgttg tgggtattcc tcacggcttg gacaactggt cgttttttgc agtttatgac 180 ggtcatgctg gatcccgagt ggcaaattac tgttcaacac atctattaga acacatcacc 240 accaatgaag acttcagggc agctgacaaa tcaggctctg ctctcgagcc ttcagtagaa 300 agtgtgaaga ctggtatcag gactggcttt ttgaaaattg atgaatatat gcgtaacttc 360 tcagacctga ggaacgggat ggacaggagc ggctcgactg cagtgggcgt gatggtctcc 420 cctacacaca tgtacttcat caactgtggt gactctcgag ctgttctgtg taggaacgga 480 caggtctgct tttctaccca ggatcacaaa ccttgtaatc cagtggagaa ggagcgcatc 540 caaaacgcag gaggcagtgt gatgatccag cgtgtgaacg gctcactagc agtgtctcgg 600 gctctggggg actatgacta caagtgtgtg gatggcaagg gccctacaga gcagcttgtt 660 tctccagagc ctgaggttta tgagattgtg agagcagaag aggatgagtt tgtcgtcttg 720 gcctgtgatg ggatctggga tgtgatgagc aatgaggagc tctgtgagtt tgttaagtct 780 aggcttgagg tgtcggacga cctggagaat gtgtgcaatt gggtagtgga cacttgttta 840 cataagggaa gtcgagataa catgagtgtt gtattagttt gcttttcaaa tgcccccaag 900 gtctcagagg aagccgtgaa gagagattca gagttggata agcacttgga atcacgggtt 960 gaagaaatca tgcagaagtc tggggaggaa ggaatgcctg atcttgccca tgtgatgcgc 1020 attttgtctg cagaaaatat cccgaattta cctcccgggg gaggtctcgc tggcaagcgc 1080 catgttattg aagctgttta tagtagactt aatccacaca aagacaatga tggg 1134 <210> 33 <211> 1368 <212> PRT <213> Artificial Sequence <220> <223> Cas9-NLS <400> 33 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45 Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp Ala Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365 <210> 34 <211> 4125 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding Cas9-NLS <400> 34 atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60 atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120 cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180 gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240 tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300 cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360 aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420 aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480 atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540 gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600 attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660 cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720 ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780 gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840 caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900 ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960 atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020 caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080 ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140 gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200 aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260 gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320 gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380 cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440 gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500 aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560 tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620 tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680 gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740 tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800 attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860 ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920 cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980 cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040 gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100 agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160 catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220 gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280 attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340 atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400 gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460 gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520 attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580 gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640 aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700 acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760 ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820 actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880 aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940 taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000 tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060 atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120 aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180 cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240 gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300 cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360 gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420 tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480 aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540 tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600 tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660 caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720 cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780 cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840 attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900 ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960 cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020 gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080 gatttgagtc agctaggagg tgaccccaag aagaagagga aggtg 4125 <210> 35 <211> 672 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding dsRed <400> 35 gcctcctccg agaacgtcat caccgagttc atgcgcttca aggtgcgcat ggagggcacc 60 gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggccac 120 aacaccgtga agctgaaggt gaccaagggc ggccccctgc ccttcgcctg ggacatcctg 180 tccccccagt tccagtacgg ctccaaggtg tacgtgaagc accccgccga catccccgac 240 tacaagaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac 300 ggcggcgtgg cgaccgtgac ccaggactcc tccctgcagg acggctgctt catctacaag 360 gtgaagttca tcggcgtgaa cttcccctcc gacggccccg tgatgcagaa gaagaccatg 420 ggctgggagg cctccaccga gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagacc 480 cacaaggccc tgaagctgaa ggacggcggc cactacctgg tggagttcaa gtccatctac 540 atggccaaga agcccgtgca gctgcccggc tactactacg tggacgccaa gctggacatc 600 acctcccaca acgaggacta caccatcgtg gagcagtacg agcgcaccga gggccgccac 660 cacctgttcc tg 672 <210> 36 <211> 708 <212> DNA <213> 人工序列 <220> <223> 编码mCherry的核酸序列 <400> 36 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaag 708 <210> 37 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Recognition site DNA sequence of sgRNA <400> 37 cccaaaagga ggatcgtact tgg 23 <210> 38 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Influenza virus HA2 <400> 38 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Trp Tyr Gly 20 <210> 39 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> KALA <400> 39 Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Ala Cys Glu Ala 20 25 30 <210> 40 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> GALA <400> 40 Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu His 1 5 10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala 20 25 30 <210> 41 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Melittin <400> 41 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 <210> 42 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Penetratin <400> 42 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 43 <211> 11 <212> PRT <213> Artificial sequence <220> <223> HIV-TAT(47-57) <400> 43 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 44 <211> 8 <212> PRT <213> Artificial sequence <220> <223> HIV-1 Rev(34-50) <400> 44 Lys Gln Ala Ile Pro Val Ala Lys 1 5 <210> 45 <211> 34 <212> PRT <213> Artificial sequence <220> <223> VP22 <400> 45 Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 1 5 10 15 Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro 20 25 30 Val Glu <210> 46 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Transportan <400> 46 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 47 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Pep-1 <220> <221> MOD_RES <222> (1)..(1) <223> ACETYLATION <220> <221> MOD_RES <222> (21)..(21) <223> AMIDATION (-NH-CH2-CH2-SH) <400> 47 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20 <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Pep-7 <400> 48 Ser Asp Leu Trp Glu Met Met Met Val Ser Leu Ala Cys Gln Tyr 1 5 10 15 <210> 49 <211> 29 <212> PRT <213> Artificial sequence <220> <223> Leucine zipper NZ <400> 49 Ala Leu Lys Lys Glu Leu Gln Ala Asn Lys Lys Glu Leu Ala Gln Leu 1 5 10 15 Lys Trp Glu Leu Gln Ala Leu Lys Lys Glu Leu Ala Gln 20 25 <210> 50 <211> 30 <212> PRT <213> Artificial sequence <220> <223> Leucine zipper CZ <400> 50 Glu Gln Leu Glu Lys Lys Leu Gln Ala Leu Glu Lys Lys Leu Ala Gln 1 5 10 15 Leu Glu Trp Lys Asn Gln Ala Leu Glu Lys Lys Leu Ala Gln 20 25 30 <210> 51 <211> 87 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding leucine zipper NZ <400> 51 gcactgaaaa aagaactgca ggcaaataaa aaagaactgg cacagctgaa atgggaactg 60 caggcactga aaaaagaact ggcacag 87 <210> 52 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding leucine zipper CZ <400> 52 gaacagctgg aaaaaaaact gcaggcactg gaaaaaaaac tggcacagct ggaatggaaa 60 aatcaggcac tggaaaaaaa actggcacag 90 <210> 53 <211> 109 <212> PRT <213> Artificial sequence <220> <223> TrxA amino acid sequence <400> 53 Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 1 5 10 15 Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 20 25 30 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 35 40 45 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 50 55 60 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 65 70 75 80 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 85 90 95 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 100 105 <210> 54 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> TrxA Nucleic Acid Sequence <400> 54 atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60 gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240 ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300 aaagagttcc tcgacgctaa cctggcc 327

Claims

1. A fusion protein, which consists of a cell-penetrating peptide, a pH-sensitive peptide, and a protease recognition sequence, wherein, The protease is selected from furin and / or cathepsin L; and, the fusion protein is composed of the cell-penetrating peptide, pH-sensitive peptide and protease recognition sequence from the N-terminus to the C-terminus; wherein, the pH-sensitive peptide can undergo conformational changes under acidic conditions to promote the fusion of the fusion protein with the endocytic vesicle membrane.

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

3. The fusion protein according to claim 1, wherein, The furin recognition sequence is as shown in SEQ ID NO:

4.

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

6.

5. The fusion protein according to claim 1, wherein, The protease recognition sequence contains a furin recognition sequence and a cathepsin L recognition sequence.

6. The fusion protein according to claim 5, wherein, The protease recognition sequence is SEQ ID NO: 4 and SEQ ID NO:

6.

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

8. The fusion protein according to claim 1, wherein, The amino acid sequence of the pH-sensitive peptide is as shown in any one of SEQ ID NOs: 8, 38-41.

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

8.

10. The fusion protein according to claim 1, wherein, The cell-penetrating peptide is selected from penetratin, Tat-derived peptide, Rev(34-50), VP22, transportan, Pep-1, Pep-7, and any combination thereof.

11. The fusion protein according to claim 1, wherein, The amino acid sequence of the cell-penetrating peptide is as shown in any one of SEQ ID NOs: 10, 42-48.

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

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

10.

14. The fusion protein according to claim 1, wherein, The protease recognition sequence is the furin recognition sequence and the cathepsin L recognition sequence from the N-terminus to the C-terminus, or the cathepsin L recognition sequence and the furin recognition sequence from the N-terminus to the C-terminus.

15. The fusion protein according to claim 1, wherein, The amino acid sequence of the fusion protein is as shown in any one of SEQ ID NOs: 12-14.

16. The fusion protein according to any one of claims 1-15, wherein, The fusion protein further comprises a specific binding sequence, and the specific binding sequence allows another molecule to specifically bind thereto.

17. The fusion protein according to claim 16, wherein, The specific binding sequence contains a leucine zipper, and the leucine zipper can form a heterodimer with its reverse sequence.

18. The fusion protein according to claim 16, wherein, The amino acid sequence of the specific binding sequence is as shown in SEQ ID NO: 49 or 50. The fusion protein according to claim 16, wherein, The specific binding sequence is located at the C-terminus of the protease recognition sequence.

20. A complex, which comprises the fusion protein according to any one of claims 1-19, and a cargo molecule.

21. The composite according to claim 20, wherein, The cargo molecule is selected from nucleic acids, peptides or proteins, saccharides, lipids, and any mixture thereof.

22. The composite according to claim 21, wherein, The nucleic acid is selected from DNA molecules, RNA molecules, antisense oligonucleotides, ribozymes, aptamers, and any combination thereof.

23. The composite according to claim 21, wherein, The nucleic acid is selected from siRNA.

24. The composite according to claim 20, wherein, The cargo molecule contains a detectable label.

25. The composite according to claim 20, wherein, The cargo molecule comprises an epitope tag, a reporter gene sequence, and / or a nuclear localization signal (NLS) sequence.

26. The composite according to claim 20, wherein, The fusion protein is fused with a cargo molecule, which is a peptide or a protein.

27. The composite according to claim 26, wherein, The cargo molecule is fused to the C-terminus of the fusion protein.

28. The composite according to claim 20, wherein, The fusion protein is chemically conjugated with a cargo molecule.

29. The composite according to claim 28, wherein, The chemical conjugation is achieved through a disulfide bond, an amide bond, a thioether bond, an ether bond, an ester bond, or a carbon-carbon bond.

30. The complex according to claim 28, wherein, The chemical conjugation is achieved through a phosphodiester bond or a phosphorothioate bond.

31. The composite according to claim 28, wherein, The cargo molecule is conjugated to the N-terminus or C-terminus of the fusion protein.

32. The composite according to claim 20, wherein, The fusion protein is non-covalently linked to a cargo molecule.

33. The complex according to claim 32, wherein, The fusion protein is the fusion protein according to any one of claims 16-19, and the cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein.

34. The complex according to claim 33, wherein, The specific binding sequence in the fusion protein comprises a leucine zipper, and the cargo molecule comprises a reverse sequence of the leucine zipper, so that the leucine zipper can form a heterodimer with the reverse sequence.

35. The composite according to claim 34, wherein, The specific binding sequence in the fusion protein is the leucine zipper NZ sequence shown in SEQ ID NO: 49, and the cargo molecule comprises the leucine zipper CZ sequence shown in SEQ ID NO: 50; or, the specific binding sequence in the fusion protein is the leucine zipper CZ sequence shown in SEQ ID NO: 50, and the cargo molecule comprises the leucine zipper NZ sequence shown in SEQ ID NO:

49.

36. The complex according to claim 32, wherein, The fusion protein and the cargo molecule are conjugated by electrostatic interaction.

37. A composition, which comprises the fusion protein according to any one of claims 1-19, and a cargo molecule.

38. The composition according to claim 37, wherein, The cargo molecule is selected from nucleic acids, peptides or proteins, saccharides, lipids, and any mixture thereof.

39. The composition according to claim 38, wherein, The nucleic acid is selected from DNA molecules, RNA molecules, antisense oligonucleotides, ribozymes, aptamers, and any combination thereof.

40. The composition according to claim 39, wherein, The nucleic acid is selected from siRNA.

41. The composition according to claim 37, which comprises the fusion protein according to any one of claims 16-19, and the cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein.

42. The composition according to claim 41, wherein, The specific binding sequence in the fusion protein comprises a leucine zipper, and the cargo molecule comprises a reverse sequence of the leucine zipper, so that the leucine zipper can form a heterodimer with the reverse sequence.

43. The composition according to claim 42, wherein, The specific binding sequence in the fusion protein is the leucine zipper NZ sequence shown in SEQ ID NO: 49, and the cargo molecule comprises the leucine zipper CZ sequence shown in SEQ ID NO: 50; or, the specific binding sequence in the fusion protein is the leucine zipper CZ sequence shown in SEQ ID NO: 50, and the cargo molecule comprises the leucine zipper NZ sequence shown in SEQ ID NO:

49.

44. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein according to any one of claims 1-19, or the complex according to claim 26 or 27, or the composition according to any one of claims 37-43.

45. A vector comprising the isolated nucleic acid molecule according to claim 44.

46. A host cell comprising the isolated nucleic acid molecule according to claim 44 or the vector according to claim 45.

47. A method for preparing the fusion protein according to any one of claims 1-19, or the complex according to claim 26 or 27, comprising culturing the host cell according to claim 46 under suitable conditions and recovering the fusion protein or the complex from the cell culture.

48. A pharmaceutical composition comprising the complex according to any one of claims 20-36, wherein, The cargo molecule is a pharmaceutically active agent or a detectable label.

49. Use of the complex according to any one of claims 20-36 or the composition according to any one of claims 37-43 as a delivery vehicle in the preparation of a medicament for treating a disease; wherein, The cargo molecule comprised in the complex or composition is a pharmaceutically active agent for treating the disease. Use according to claim 49, wherein, The disease is a disease associated with necroptosis, and the cargo molecule comprises protein phosphatase 1B; wherein the disease associated with necroptosis includes liver injury, inflammatory diseases, ischemia-reperfusion injury, and / or neurodegenerative diseases.

51. A kit comprising the fusion protein according to any one of claims 1-19, the complex according to any one of claims 20-36, the composition according to any one of claims 37-43, the isolated nucleic acid molecule according to claim 44, the vector according to claim 45, or the host cell according to claim 46; the kit further comprises instructions for transfection and / or intracellular delivery.

52. Use of the fusion protein according to any one of claims 1-19, the complex according to any one of claims 20-36, the composition according to any one of claims 37-43, the isolated nucleic acid molecule according to claim 44, the vector according to claim 45, or the host cell according to claim 46 as a delivery reagent for non-diagnostic therapeutic purposes.

53. A method for delivering a cargo molecule into a cell for non-diagnostic therapeutic purposes, which comprises contacting the cell with the complex according to any one of claims 20-36, wherein the complex comprises the cargo molecule; wherein, The contact between the cell and the complex is carried out in vitro.

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