WO2021004539A1 - 用于胞内递送分子的复合物 - Google Patents

用于胞内递送分子的复合物 Download PDF

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WO2021004539A1
WO2021004539A1 PCT/CN2020/101424 CN2020101424W WO2021004539A1 WO 2021004539 A1 WO2021004539 A1 WO 2021004539A1 CN 2020101424 W CN2020101424 W CN 2020101424W WO 2021004539 A1 WO2021004539 A1 WO 2021004539A1
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Prior art keywords
sequence
fusion protein
complex
cargo molecule
seq
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PCT/CN2020/101424
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English (en)
French (fr)
Inventor
葛胜祥
于思远
杨晗
潘海峰
任书玲
李廷栋
郭清顺
熊君辉
张军
夏宁邵
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厦门大学
厦门万泰沧海生物技术有限公司
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Application filed by 厦门大学, 厦门万泰沧海生物技术有限公司 filed Critical 厦门大学
Priority to KR1020227004290A priority Critical patent/KR20220034182A/ko
Priority to EP20837897.6A priority patent/EP4006057A4/en
Priority to AU2020310380A priority patent/AU2020310380A1/en
Priority to JP2022501316A priority patent/JP2022549057A/ja
Priority to US17/626,351 priority patent/US20220265837A1/en
Publication of WO2021004539A1 publication Critical patent/WO2021004539A1/zh

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Definitions

  • the present invention relates to the field of molecular biology, and more specifically, to fusion proteins and complexes for intracellular delivery of cargo molecules.
  • the cell membrane as a selective permeability barrier is essential for cell survival and function. Although small molecules can pass through the cell membrane through the natural process of the cell or the direct diffusion of the lipid bilayer, in most cases, exogenous The effective passage of intracellular loads such as active biological macromolecules from the plasma membrane of the cell is still the main obstacle to the cell transport process. Therefore, a molecular transport tool that can effectively improve the transport efficiency of intracellular loads to living cells is extremely important for its applications in biomedicine and other fields.
  • CPPs Cell-penetrating peptides
  • Cell penetrating peptides usually contain 5-30 amino acids, which can carry biological macromolecules through the cell membrane and into the cell by 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 for intracellular delivery of intracellular loads.
  • CPPs cationic, rich Contains arginine and lysine residues and has a strong positive charge at physiological pH
  • amphipathic including polar and non-polar regions, except for lysine and arginine distributed throughout the sequence
  • hydrophobic residues such as valine, leucine, isoleucine and alanine
  • hydrophobic type mainly containing non-polar amino acids.
  • CPPs interact with negatively charged or hydrophobic lipid bilayers on the cell membrane surface through their strong positive or hydrophobic groups. When carrying small molecules, they can directly translocate across the cell membrane in a non-energy-consuming way.
  • CPP When carrying biological macromolecules, basically Use energy-dependent endocytosis (endocytosis) into the cell. Due to the advantages of low dosage of CPPs when introduced into cells, time required for transport, controllable dosage, simple operation and low toxic and side effects, CPP has been widely used to transport polypeptides, proteins, and oligonucleotides in vitro and in vivo. , Plasmids, liposomes and metal ions, small molecule fluorescence, nanoparticles, etc.
  • CPPs have many advantages in transducing biological macromolecules, they still have some limitations.
  • the first thing that needs to be solved is its low transmembrane delivery efficiency.
  • CPPs interact with the cell membrane surface when transducing the internal load into the cell, and then enter the cell through endocytosis.
  • Cell endocytosis can be divided into four main ways according to its endocytosis mechanism: macropinocytosis, clathrin-mediated endocytosis, caveolin/lipid raft mediated Caveolae/lipid raft-mediated endocytosis and clathrin/caveolae-independent endocytosis (clathrin/caveolae-independent endocytosis).
  • the main strategy is to destroy the integrity of the vesicle vesicle membrane during the process of maturation and acidification, so that its contents (including the intracellular load to be delivered) are released into the cytoplasm.
  • a buffer such as chloroquine, methylamine, or ammonium chloride can be added to the system to physically promote the penetration and rupture of endocytic vesicles, but because of its strong Its cytotoxicity hinders its clinical application.
  • the most effective way is to use pH sensitive fusogenic peptides derived from viruses, bacteria, animals, plants or humans.
  • the pH-sensitive peptide contains a certain proportion of hydrophobic amino acids, and the conformation changes drastically at low pH.
  • CPPs mediated cell endocytosis
  • the endocytic vesicles are in the process of maturation and acidification.
  • the pH-sensitive peptides coupled with CPPs undergo conformational changes and bind to the lipid bilayers of the vesicle membrane As a result, the integrity of the phospholipid bilayer membrane is severely disturbed, small pores are formed on it or the capsule membrane is ruptured, and finally the transported biological macromolecules are released into the cytoplasm.
  • pH-sensitive peptides are a peptide from the stem region of influenza virus hemagglutinin antigen (hereinafter referred to as HA2), and INF7, which is artificially modified based on HA2, has lower toxicity and better membrane rupture effect. Both are related to CPPs and proteins. After molecular fusion expression, the escape efficiency of endocytic vesicles of transporter molecules can be improved.
  • the inventor of the present application unexpectedly discovered that the combination of pH-sensitive peptides and specific protease recognition sequences as delivery vehicles can significantly increase the release of cargo molecules from endocytic vesicles, thereby significantly improving the cytoplasmic delivery of cargo molecules Efficiency, so that the cargo molecules can give full play to their corresponding biological functions. Based on these findings, the present inventors have developed a carrier system capable of achieving efficient cytoplasmic delivery.
  • the 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.
  • the furin recognition sequence comprises or consists of the following sequence: RX 1 -X 2 -R ⁇ (SEQ ID NO: 1), wherein X 1 is any amino acid and X 2 is K or R, ⁇ indicates the cutting site.
  • the furin recognition sequence comprises or consists of the following sequence: RRX 1 -X 2 -R ⁇ (SEQ ID NO: 2).
  • X 1 is selected from alanine (A), arginine (R), aspartic acid (D), cysteine (C), glutamine (Q), glutamine Acid (E), histidine (H), isoleucine (I), glycine (G), aspartic acid (N), leucine (L), lysine (K), methionine Acid (M), Phenylalanine (F), Coine (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y) and Valine ( V).
  • the furin recognition sequence comprises or consists of the following sequence: RRHkr ⁇ (SEQ ID NO: 3).
  • the furin recognition sequence comprises or consists of the following sequence: QSVASSRRHKR ⁇ FAGV (SEQ ID NO: 4).
  • the lysosomal cysteine protease is selected from cathepsin B, cathepsin C, cathepsin X, cathepsin S, cathepsin L, cathepsin D, or cathepsin H.
  • the lysosomal cysteine protease is cathepsin L.
  • the cathepsin L recognition sequence comprises or consists of the following sequence: NNTHDLVGDVRLAGV (SEQ ID NO: 6).
  • the protease recognition sequence comprises a furin recognition sequence and a cathepsin L recognition sequence.
  • the protease recognition sequence is a single-chain polypeptide, which includes a furin recognition sequence and a cathepsin L recognition sequence from N-terminus to C-terminus, or it includes a cathepsin L recognition sequence from N-terminus to C-terminus Sequence and Furin recognition sequence.
  • 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.
  • pH-sensitive fusogenic peptide and “pH-sensitive peptide” are used interchangeably, which refers to a class that can undergo conformational changes under acidic conditions (for example, pH ⁇ 6.5) Thereby promoting the fusion of the polypeptide with the endocytic vesicle membrane.
  • acidic conditions for example, pH ⁇ 6.5
  • pH-sensitive peptides are endocytosed by cells, the endocytic vesicles are in the process of maturation and acidification. Once the pH drops to the critical point, such peptides undergo conformational changes and bind to the lipid bilayers of the vesicle membrane to violently disturb the phospholipids.
  • the integrity of the double-layer membrane can form small pores or cause the lysis of the capsule membrane, thereby releasing the transported biological macromolecules into the cytoplasm.
  • polypeptides are well known in the art and are described in, for example, Varkouhi, Amir K., et al. Journal of Controlled Release 151.3 (2011): 220-228; Erzo-Oliveras A, Muthukrishnan N, Baker R, et al. Pharmaceuticals, 2012, 5(11): 1177-1209, all of which are incorporated herein by reference.
  • Viral protein source HA2 (influenza virus) and its mutants KALA, GALA; penton base (adenovirus or rhinovirus), gp41 (HIV), L2 (papilloma virus), envelope protein (Western Nile virus (West Nile virus);
  • Bacterial protein sources Listeriolysin O (LLO), Pneumococcal pneumolysin (PLO), Streptococcal streptolysin O (SLO), Diphtheria toxin ( Diphtheria toxin, Pseudomonas aeruginosa exotoxin A (Shiga toxin), Cholera toxin (Cholera toxin);
  • Plant protein sources Ricin, Saporin, Gelonin toxin;
  • Human/animal protein sources human calcitonin, fibroblast growth factor receptor, melittin (Melittin);
  • 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.
  • the mutant of the influenza virus HA2 is selected from INF7 (SEQ ID NO: 8), KALA (SEQ ID NO: 39) or GALA (SEQ ID NO: 40).
  • the pH-sensitive fusion peptide comprises INF7. In some embodiments, the pH-sensitive fusion peptide comprises or consists of the following sequence: SEQ ID NO: 8.
  • cell penetrating peptide is also called “cell penetrating peptide", “protein translocation domain (PTD), "Trojan horse peptides” or “transduction peptide”.
  • Transduction peptide and the like refer to polypeptides that can promote cellular uptake of various molecules (for example, various macromolecules including proteins or nucleic acids). Such polypeptides are well known in the art and are described in, for example, Stewart KM, et al. Org Biomol Chem.
  • the CPP that can be used in the fusion protein of the present invention includes but is not limited to: cationic: Penetratin, HIV-TAT-47-57, HIV-1 Rev 34-50, FHV coat-35-49, Oligoarginines (R9-R12), CCMV Gag-7-25, S413-PV, VP22, BP16, DPV3, DPV6, FAH coat protein, Protamine 1 (Protamine 1), human cJun, Engrailed-2, Islet-1, HoxA -13, TP10, etc.; amphipathic type: transportan, Transportan 10, Pep-1, MPG ⁇ , MPG ⁇ , CADY, Pepfect6, Pepfect14, Pepfect15, NickFect, Hel, sC18, pVEC, ARF(1-22) , YTA2, PAR1 (Palmitoyl-SFLLRN), F2Pal10 (Palmitoyl-SFLLRN), BprPp (1-30),
  • the CPP used in the fusion protein of the present invention can also be selected from polypeptide sequences that have about 60, 70, 80, 90, 95, 99%, or 100% sequence identity with any polypeptide sequence as described above, as long as The polypeptide sequence still retains its biological activity, that is, it promotes the cellular uptake of the molecule.
  • the cell penetrating peptide is selected from the group consisting of Penetratin (SEQ ID NO: 42), Tat derived peptide, Rev(34-50) (SEQ ID NO: 44), VP22 (SEQ ID NO: 44), ID NO: 45), transportan (SEQ ID NO: 46), Pep-1 (SEQ ID NO: 47), Pep-7 (SEQ ID NO: 48), and any combination thereof.
  • the Tat-derived peptide is selected from Tat (48-60) (SEQ ID NO: 10) or Tat (47-57) (SEQ ID NO: 43).
  • the cell-penetrating peptide comprises a Tat-derived peptide, such as Tat(48-60).
  • the cell penetrating peptide comprises or consists of the following sequence: SEQ ID NO: 10.
  • the fusion protein of the present invention includes the pH-sensitive fusion peptide, cell penetrating peptide, and protease recognition sequence from N-terminus to C-terminus.
  • the fusion protein comprises the pH-sensitive fusion peptide, cell-penetrating peptide, furin recognition sequence, and cathepsin L recognition sequence from N-terminus to C-terminus.
  • the fusion protein includes the pH-sensitive fusion peptide, cell penetrating peptide, cathepsin L recognition sequence, and furin recognition sequence from N-terminus to C-terminus.
  • the fusion protein of the present invention includes the cell-penetrating peptide, pH-sensitive fusion peptide and protease recognition sequence from N-terminus to C-terminus.
  • the fusion protein includes the cell penetrating peptide, pH-sensitive fusion peptide, furin recognition sequence, and cathepsin L recognition sequence from N-terminus to C-terminus.
  • the fusion protein comprises the cell penetrating peptide, pH-sensitive fusion peptide, cathepsin L recognition sequence, and furin recognition sequence from N-terminus to C-terminus.
  • the fusion protein comprises or consists of a sequence selected from the following: SEQ ID NOs: any one of 12-14.
  • the fusion protein may further include a protein tag at its N-terminus.
  • the protein tag has a solubilizing effect.
  • the protein tag is selected from TrxA, SUMO, NusA, MBP, GST.
  • the second aspect of the present invention also provides a fusion protein, which, on the basis of the fusion protein of the first aspect, further comprises a specific binding sequence that allows additional molecules (such as polypeptides, proteins or nucleic acids) (Such as DNA)) specifically bind to it.
  • additional molecules such as polypeptides, proteins or nucleic acids
  • specific binding refers to a non-random binding reaction between two molecules, such as a reaction between an antibody (or an antigen-binding fragment thereof) and the antigen (or epitope) against which it is directed, or two A heterodimer reaction between two amino acid sequences (for example, two leucine zipper domains in antiparallel).
  • the specific binding sequence comprises a leucine zipper, and the leucine zipper can form a heterodimer with its reverse sequence.
  • the specific binding sequence comprises a leucine zipper NZ or CZ. It is known in the art that the leucine zipper NZ and CZ are reverse sequences of each other, and there is a strong interaction between the two, so that the antiparallel leucine zipper NZ and CZ can form a heterodimer.
  • the leucine zipper NZ comprises the sequence shown in SEQ ID NO:49.
  • the leucine zipper CZ comprises the sequence shown in SEQ ID NO:50.
  • 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.
  • the specific binding sequence is located at the C-terminus of the protease recognition sequence. In certain embodiments, the fusion protein includes the specific binding sequence at its C-terminus.
  • the fusion protein may further include a protein tag at its N-terminus.
  • the protein tag has a solubilizing effect.
  • the protein tag is selected from TrxA, SUMO, NusA, MBP, GST.
  • the fusion protein of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods (recombinant technology), or by chemical synthesis methods (for example, Fmoc solid phase method).
  • the fusion protein of the present invention is not limited by its production method.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of the first or second aspect of the invention.
  • the present invention provides a vector (such as a cloning vector or an expression vector), which comprises the isolated nucleic acid molecule as described above.
  • a vector such as a cloning vector or an expression vector
  • the vector is, for example, a plasmid, cosmid, phage, and the like.
  • the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • a method of preparing the fusion protein of the first or second aspect of the present invention which comprises culturing the host cell as described above under conditions that allow the expression of the fusion protein, and obtaining the cultured host cell The fusion protein is recovered from the culture.
  • the present invention provides a complex comprising the fusion protein of the first or second aspect of the present invention and a cargo molecule (cargo).
  • the cargo molecule can be any molecule.
  • the cargo molecule is selected from nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds, and any mixtures thereof.
  • the nucleic acid is selected from DNA molecules, RNA molecules, siRNA, antisense oligonucleotides, ribozymes, aptamers, and any combination thereof.
  • the molecular weight of the cargo molecule is less than 10000 Da, for example, less than 5000 Da, less than 3000 Da, or less than 1000 Da.
  • the cargo molecule contains a detectable label, such as an enzyme, a radionuclide, a fluorescent dye, a chemiluminescent substance, or biotin.
  • a detectable label such as an enzyme, a radionuclide, a fluorescent dye, a chemiluminescent substance, or biotin.
  • the cargo molecule comprises an epitope tag, a reporter gene sequence, and/or a nuclear localization signal (NLS) sequence.
  • the cargo molecule is a peptide or protein.
  • Epitope tags that can be used in cargo molecules are well known to those skilled in the art, examples of which include but are not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to The purpose (e.g., purification, detection or tracing) selects the appropriate epitope tag.
  • the cargo molecule comprises a His tag.
  • Reporter gene sequences that can be used in cargo molecules are well known to those skilled in the art, examples of which include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, etc.
  • 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 SV40 virus large T antigen. In certain exemplary embodiments, the NLS sequence is shown in SEQ ID NO: 15.
  • the fusion protein of the present invention is fused to a cargo molecule, wherein the cargo molecule is a peptide or protein.
  • the fusion protein is as defined in the first aspect.
  • 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.
  • the complex of the present invention comprises a single-chain polypeptide comprising from N-terminus to C-terminus: the pH-sensitive fusion peptide, cell-penetrating peptide, protease recognition sequence, and cargo molecule.
  • the single-chain polypeptide comprises the pH-sensitive fusion peptide, cell-penetrating peptide, furin recognition sequence, cathepsin L recognition sequence, and cargo molecules from N-terminus to C-terminus.
  • the single-chain polypeptide comprises the pH-sensitive fusion peptide, cell-penetrating peptide, cathepsin L recognition sequence, furin recognition sequence, and cargo molecule from N-terminus to C-terminus.
  • the complex of the present invention comprises a single-chain polypeptide, which comprises from N-terminus to C-terminus: the cell penetrating peptide, pH-sensitive fusion peptide, protease recognition sequence and cargo molecule.
  • the single-chain polypeptide comprises the cell-penetrating peptide, pH-sensitive fusion peptide, furin recognition sequence, cathepsin L recognition sequence, and cargo molecule from N-terminus to C-terminus.
  • the single-chain polypeptide comprises the cell-penetrating peptide, pH-sensitive fusion peptide, cathepsin L recognition sequence, furin recognition sequence, and cargo molecule from N-terminus to C-terminus.
  • the cargo molecule is a zinc finger protein (such as ZFP9), a protein phosphatase (such as Ppm1b), or a Cas effector protein (such as Cas9).
  • ZFP9 zinc finger protein
  • Ppm1b protein phosphatase
  • Cas effector protein such as Cas9.
  • the expression "Cas effector protein” refers to the effector protein of the CRISPR-Cas system.
  • the zinc finger protein or Cas effector protein comprises an NLS sequence.
  • the fusion protein of the present invention is chemically coupled with cargo molecules.
  • the "chemical coupling” refers to the bond obtained in the chemical reaction between the reactive group contained in the fusion protein and the reactive group contained in the cargo molecule. After the chemical reaction, the two parts are connected by a covalent bond .
  • the fusion protein, the cargo molecule, or both can be modified in a separate reaction with a linker molecule so that they respectively contain the reactive groups required for the chemical coupling.
  • linker molecule used to modify the fusion protein or cargo molecule depends on the coupling strategy used.
  • the fusion protein is as defined in the first aspect.
  • 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.
  • 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.
  • the cargo molecule is a nucleic acid.
  • the fusion protein of the invention is non-covalently linked to the cargo molecule.
  • the fusion protein is conjugated to the cargo molecule through electrostatic interaction.
  • the cargo molecule is a nucleic acid.
  • the fusion protein is as defined in the second aspect, and the fusion protein is non-covalently linked to the cargo molecule through the specific binding sequence it contains.
  • the cargo molecule comprises a domain capable of specifically binding to the specific binding sequence in the fusion protein.
  • the domain capable of specifically binding to the specific binding sequence in the fusion protein is an amino acid sequence.
  • the cargo molecule is a peptide or protein. In certain embodiments, the cargo molecule includes at its N-terminus the amino acid sequence capable of specifically binding to the specific binding sequence in the fusion protein.
  • the specific binding sequence in the fusion protein comprises a leucine zipper
  • the cargo molecule comprises the reverse sequence of the leucine zipper, so that the leucine zipper can interact with the reverse sequence of the leucine zipper.
  • a heterodimer is formed toward the sequence.
  • the specific binding sequence in the fusion protein comprises a leucine zipper NZ (for example, the sequence shown in SEQ ID NO: 49), and the cargo molecule comprises a leucine zipper CZ (for example, SEQ ID NO: 50).
  • the specific binding sequence in the fusion protein comprises a leucine zipper CZ (for example, the sequence shown in SEQ ID NO: 50), and the cargo molecule comprises a leucine zipper NZ (for example, SEQ ID NO: 49).
  • the complex of the present invention can be prepared by various methods known in the art, for example, by genetic engineering methods (recombinant technology), or by chemical synthesis methods (for example, Fmoc solid phase method).
  • the complex of the present invention is not limited by its production method.
  • the complex of the present invention can be obtained by genetic engineering recombination technology.
  • a DNA molecule encoding the complex is obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into the expression vector and then transfected into the host cell. Then, the transfected host cell is cultured under specific conditions, and the complex of the present invention is expressed.
  • the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a complex as described above.
  • the present invention provides a vector (such as a cloning vector or an expression vector), which comprises the isolated nucleic acid molecule as described above.
  • a vector such as a cloning vector or an expression vector
  • the vector is, for example, a plasmid, cosmid, phage, and the like.
  • the present invention provides a host cell comprising the isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • a method for preparing the complex as described above which comprises culturing the host cell as described above under conditions that allow the expression of the complex, and recovering the complex from the cultured host cell.
  • the complex comprises culturing the host cell as described above under conditions that allow the expression of the complex, and recovering the complex from the cultured host cell.
  • the complex of the present invention can be obtained by the following exemplary method: after allowing the fusion protein and the cargo molecule to be contained separately The fusion protein is mixed with the cargo molecule under the condition that the reactive group is chemically reacted, so that the two parts are connected by a covalent bond.
  • the method further includes: using a linker molecule to modify the fusion protein, the cargo molecule, or both so that they each contain the reactive groups required for the above chemical reaction.
  • the cargo molecule is a nucleic acid.
  • the complex of the present invention when the complex comprises a fusion protein and a cargo molecule conjugated by electrostatic interaction, can be obtained by the following exemplary method: (1) The fusion protein of the present invention is combined with The cargo molecules are mixed to form a mixture; and (2) the mixture is incubated so that the fusion protein and the cargo molecules form a complex.
  • the cargo molecule is a nucleic acid.
  • the complex of the present invention when the complex includes a fusion protein and a cargo molecule that are specifically bound rather than covalently linked, can be obtained by the following exemplary methods: (1) Make the present invention The fusion protein of the second aspect is mixed with a cargo molecule, the cargo molecule comprising a domain that specifically binds to a specific binding sequence in the fusion protein; and (2) incubating the mixture to make the fusion The protein forms a complex with the cargo molecule.
  • the cargo molecule is a polypeptide or protein.
  • the present invention also provides a composition comprising the fusion protein of the present invention and a cargo molecule.
  • the cargo molecule is selected from nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds, and any mixtures thereof.
  • the nucleic acid is selected from DNA molecules, RNA molecules, siRNA, antisense oligonucleotides, ribozymes, aptamers, and any combination thereof.
  • the cargo molecule is selected from nucleic acids.
  • the cargo molecule is a polypeptide or protein.
  • the fusion protein is as defined in the second aspect.
  • the cargo molecule includes a domain capable of specifically binding to the specific binding sequence in the fusion protein.
  • the fusion protein of the present invention can efficiently release cargo molecules from endocytic vesicles. Once cargo molecules are available in the cytoplasm, they can play any role related to them. Therefore, the fusion protein of the present invention can be used as an intracellular delivery agent, which can be further used in research and therapeutic and diagnostic applications.
  • the present invention provides a pharmaceutical composition which contains the fusion protein, complex, composition, isolated nucleic acid molecule, vector or host cell of the present invention, and a pharmaceutically acceptable carrier and/ Or excipients.
  • the cargo molecule contained in the complex or composition is a pharmaceutically active agent.
  • the cargo molecule contained in the complex or composition is a detectable label.
  • the label can be used for diagnosis, for research drug treatment (for example, absorption, distribution, metabolism, excretion), for research treatment or drug efficacy or side effects, etc.
  • the present invention also relates to the fusion protein of the present invention, an isolated nucleic acid molecule, vector or host cell comprising a nucleotide sequence encoding the fusion protein, as a delivery agent (for example, an intracellular delivery agent and/or transfection agent).
  • a delivery agent for example, an intracellular delivery agent and/or transfection agent.
  • Dyeing reagent is used to prepare medicine.
  • the present invention also relates to 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, which is prepared for use in the treatment of diseases.
  • the disease is a disease related to programmed cell death, wherein the cargo molecule comprises protein phosphatase 1B.
  • the diseases associated with programmed cell necrosis include liver injury (eg, drug-induced liver injury), inflammatory diseases, ischemia-reperfusion injury, and/or neurodegenerative diseases.
  • 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 a tablet, pill, suspension, emulsion, solution, gel, capsule, Powders, granules, elixirs, lozenges, suppositories, injections (including injections, freeze-dried powders), inhalants, sprays and other forms.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • 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.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs and the like.
  • the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl acetate, ethyl acetate, benzyl alcohol, benzyl benzoate Esters, propylene glycol, 1,3-butanediol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene Fatty acid esters of glycols and sorbitan and mixtures thereof.
  • inert diluents commonly used in the art, such as water or other solvents, solubilizers and
  • liquid dosage forms for oral administration may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and fragrances.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and fragrances.
  • Solid dosage forms for oral administration include capsules, tablets, pills, lozenges, powders, granules and the like.
  • the solid dosage form 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 acacia); wetting agents (such as cetyl alcohol and glyceryl monostearate); disintegrating agents (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, laurel Sodium sulfate); and mixtures thereof.
  • fillers such as lactose, sucrose, glucose, mannitol, starch, microcrystalline cellulose, galactose, crospovidone And calcium
  • the fusion protein, complex or composition, or pharmaceutical composition of the present invention can also be administered by non-oral route.
  • parenteral administration for example, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, and injection.
  • the dosage form for parenteral administration may be an injection preparation, including injection, sterile powder for injection, or concentrated solution for injection.
  • the injection dosage form 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 agent.
  • transdermal administration such as via a transdermal patch or iontophoresis device
  • intraocular administration or intranasal or inhalation administration.
  • the dosage forms for transdermal administration can be topical gels, sprays, ointments and creams.
  • topical dosage forms may contain ingredients that enhance the absorption or penetration of the active compound through the skin or other areas of action.
  • the dosage form for rectal administration may be a suppository.
  • fusion protein, complex or composition, or pharmaceutical composition of the present invention can be prepared by any known pharmaceutical process, such as effective formulation and administration method.
  • the present invention provides a kit containing the fusion protein, complex, composition, isolated nucleic acid molecule, vector or host cell of the present invention.
  • the kit further includes instructions for transfection and/or intracellular delivery.
  • the kit is used for transfection and/or intracellular delivery of cargo molecules (e.g., nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds, and any mixtures thereof).
  • the cell is a mammalian cell, such as a human cell.
  • 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 agent (for example, a transfection agent or an intracellular delivery agent).
  • a delivery agent for example, a transfection agent or an intracellular delivery agent.
  • the delivery reagent is used to deliver cargo molecules (e.g., nucleic acids, peptides or proteins, carbohydrates, lipids, chemical compounds, and any mixtures thereof) intracellularly.
  • the cell is a mammalian cell, such as a human cell.
  • the invention provides a method for delivering a molecule into a cell, which comprises contacting the cell with a complex of the invention, wherein the complex includes the molecule.
  • the contact of the cell with the complex is performed in vivo.
  • the contact of the cell with the complex is performed ex vivo.
  • the contacting of the cell with the complex is performed in vitro.
  • the cell is a eukaryotic cell, such as a mammalian cell, such as a human cell.
  • isolated refers to obtained from the natural state by artificial means. If a certain "isolated” substance or component appears 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. For example, for a certain unisolated polynucleotide or polypeptide that is naturally present in a living animal, the same polynucleotide or polypeptide with high purity isolated from this natural state is called It is "separated”.
  • isolated does not exclude the presence of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 fruit fly cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 fruit fly cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a certain position in the two sequences to be compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • each molecule is the same 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 to be compared ⁇ 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match).
  • the comparison is made when two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput.
  • 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, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, ionic strength enhancers, Agents that maintain osmotic pressure, agents that delay absorption, diluents, adjuvants, preservatives, stabilizers, etc.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include but are not limited to cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents for maintaining osmotic pressure include but are not limited to sugar, NaCl and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol) and the like.
  • Adjuvants include, but are not limited to, aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant) and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, paraben, chlorobutanol, phenol, sorbic acid and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug (for example, the inhibitory activity of PSD-95 ubiquitination), including but not limited to sodium glutamate, gelatin, SPGA, Sugars (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) and so on.
  • the active ingredient in the drug for example, the inhibitory activity of PSD-95 ubiquitination
  • the desired activity of the active ingredient in the drug for example, the inhibitory activity of PSD-95 ubiquitination
  • the desired activity of the active ingredient in the drug for example, the inhibitory activity of PSD-95 ubiquitination
  • the desired activity of the active ingredient in the drug for example, the inhibitory activity of PSD-95 ubiquit
  • treatment refers to a method implemented in order to obtain beneficial or desired clinical results.
  • beneficial or desired clinical results include (but are not limited to) alleviation of symptoms, reduction of the scope of the disease, stabilization (ie, no worsening) of the disease state, delay or slowdown of the development of the disease, improvement or alleviation of the disease The state and relief of symptoms (whether partial or full), whether detectable or undetectable.
  • treatment can also refer to prolonging survival compared to expected survival (if not receiving treatment).
  • the term "subject" refers to a mammal, such as a primate mammal, such as a human.
  • the delivery system of the present invention can significantly improve the release of cargo molecules from endocytic vesicles, thereby significantly improving the cytoplasmic delivery efficiency of cargo molecules, so that the cargo molecules can 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, can be used in many fields such as research, therapy, diagnosis, and has broad application prospects and clinical value.
  • Figure 1 shows the principle schematic diagram of the Split-GFP system in Example 1 for detecting the escape efficiency of endocytic vesicles.
  • Figure 2 shows a schematic diagram of the structure of the delivery system-GFP ⁇ 1-10 protein complex in Example 1.
  • Figure 3-4 shows the results of SDS-PAGE of the delivery system-GFP ⁇ 1-10 complex in Example 1.
  • Figure 5 shows the results of flow cytometry analysis of GFP ⁇ 1-10 transduced by the delivery system in Example 1.
  • Figure 6 shows the results of fluorescence microscopy observations of GFP ⁇ 1-10 transduced by the delivery system in Example 1.
  • Figure 7 shows the results of flow cytometry analysis of GFP ⁇ 1-10 transduced by the delivery system containing restriction site mutations in Example 1.
  • Figure 8 shows the relative proportions of cleaved and uncut/complete GFP ⁇ 1-10 in the cell after transduction by the delivery system in Example 1, and the Western blot test results of the retention time in the cell.
  • Figure 9 shows the Western blot detection results of the relative proportions of cleaved and uncut/complete GFP ⁇ 1-10 in the cell after transduction by the delivery system containing the restriction site mutation in Example 1.
  • Figure 10 shows the results of flow cytometry analysis after transduction of the delivery system containing restriction enzyme cleavage sites but no pH-sensitive peptides in Example 1.
  • Figure 11 shows the Western blot detection results of the relative proportions of cleaved and uncut/complete GFP ⁇ 1-10 in the cell after transduction of the delivery system containing the restriction site but no pH-sensitive peptide in Example 1.
  • Figure 12 shows a schematic diagram of the structure of the delivery system-ZFP9 complex in Example 2.
  • Figure 13 shows the SDS-PAGE results of the delivery system-ZFP9 complex in Example 2.
  • Figure 14 shows the map of the eukaryotic expression plasmid containing the ZFP9 binding site in Example 2.
  • Figures 15-16 show the results of flow cytometry analysis of ZFP9 transduced by the delivery system in Example 2.
  • Figure 17 shows a schematic diagram of the structure of the delivery system-Ppm1b complex in Example 3.
  • Figure 18 shows the results of SDS-PAGE of the delivery system-Ppm1b complex in Example 3.
  • Example 20 shows a schematic diagram of the structure of the delivery system-Cas9 complex in Example 4.
  • FIG. 21 shows the SDS-PAGE results of the delivery system-Cas9 complex in Example 4.
  • Figure 22 shows a schematic diagram of the principle of using HEK293T-RFP reporter cells to detect the editing efficiency of CRISPR/Cas9 in Example 4.
  • Figure 23 shows the RFP repoter lentiviral plasmid map in Example 4.
  • Figure 24 shows the results of flow cytometry analysis of the gene editing efficiency of the delivery system-Cas9 complex in Example 4.
  • FIG. 25 shows the principle of the delivery system based on the adapter method in Embodiment 5.
  • Figure 26 shows the design of recombinant protein related clones in the delivery system based on the adapter method in Example 5.
  • Figure 27 shows the results of purification of recombinant protein in the delivery system based on the adapter method in Example 5.
  • Fig. 28 shows the evaluation of the delivery effect of the adapter-based delivery system based on the Split-GFP vesicle escape system in Example 5.
  • Furin recognition sequence-1 Furin recognition sequence-2 3 Furin recognition sequence-3 4 Furin recognition sequence-4 (Ne) 5 Nucleic acid sequence encoding Ne 6 CTSL recognition sequence N 7 Nucleic acid sequence encoding N 8 INF7 9 Nucleic acid sequence encoding INF7 10 Tat(48-60) 11 Nucleic acid sequence encoding Tat (48-60) 12 Fusion protein TIN 13 Fusion Protein TINe 14 Fusion protein TINNe 15 NLS 16 CTSL recognition sequence Na 17 CTSL recognition sequence Nb 18 Furin recognition sequence Nc 19 Furin recognition sequence Nd 20 CTSD identification sequence Nf twenty one Mutant N twenty two Mutant Ne
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FMAusubel et al., Compiled Molecular Biology Experiment Guide, 3rd edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer.
  • the embodiments describe the present invention by way of example, and are not intended to limit the scope of protection claimed by the present invention.
  • DNA polymerase (TaKaRa, R040A), DNA recovery kit (TianGen, DP214-03), plasmid small extraction kit (TianGen, DP103-03), plasmid large extraction kit (QIAGEN) ,12663), 5 tubes of Gibson assembly premix (NEB, E2611L), DNA marker (ThmeroFisher, SM0331), agarose (Biowest, BW-R0100),
  • the materials required for large-scale protein expression are as follows: peptone (BiSIGMA-ALDRICH, T7293-1KG), yeast powder (OXOID, LP0021B), sodium chloride (Xilong Chemical Industry, 10011012AR), IPTG (Inalco, 1758-1400)
  • 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)
  • glycerol/glycerol/C 3 H 8 O 3 SIGMA-ALDRICH, G5516
  • KCl Xilong Chemical Industry, 1002007
  • Na 2 HPO 4 ⁇ 12H 2 O Xilong Chemical Industry
  • KH 2 PO 4 Xilong Chemical Industry, 1002048AR500
  • Imidazole SIGMA-ALDRICH, V900153
  • Tris base Seebio, 183995
  • Glucose Xilong Chemical Industry, 1064008AR500
  • BCA protein concentration determination kit Thermo Scientific, 23227
  • FBS FBS (GIBCO, 10099-133), DMEM (GIBCO, 11965092), trypsin (AMRESCO, 0458);
  • lentivirus packaging plasmid 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);
  • the GFP ⁇ 1-10, ZFP9, Ppm1b, dsRed, mCherry, and Histone-H3 related plasmids used in the experiment were all synthesized by the company (Biotech) and used to amplify the Cas9 sequence plasmid pCasKP-hph (Addgene, 117232);
  • TNF- ⁇ Novoprotein, CF09
  • PI ThimeroFisher, P3566
  • HEK-293T human renal epithelial cells
  • L929 mouse fibroblasts
  • Example 1 Evaluation of the endocytic vesicle escape efficiency of the delivery system based on the Split-GFP system
  • the 11 ⁇ sheets of GFP are split into large fragments ( ⁇ 1-10) and small fragments ( ⁇ 11), both of which lose their fluorescent activity, but if they meet, they can associate spontaneously and restore GFP The fluorescence performance.
  • HEK293T cells stably expressing Histone- ⁇ 11, and at the same time fused GFP ⁇ 1-10 with nuclear import signal (NLS) as the Cargo and expressed in the cell delivery system to be evaluated, and then transduced the stable cell line.
  • NLS nuclear import signal
  • Nucleic acid sequence and connect the parts through multiple rounds of PCR, and introduce the NdeI restriction site and the NdeI restriction site corresponding to pET21b(+) at the 5'end of the fragment through the upstream guide in the last round of PCR
  • the BamHI restriction site and the overlapping sequence downstream of the BamHI restriction site corresponding to pET-21b(+) are introduced at the 3'end of the fragment through the downstream guide.
  • the pET-21b(+) plasmid was digested with NdeI and BamHI.
  • the insert with overlapping sequence was ligated to the digestion vector pET-21b(+) by GIBSON assembly.
  • the coding sequence of Histone-H3 (SEQ ID NO: 25) was amplified by PCR, and the GFP ⁇ 11 coding sequence (SEQ ID NO: 26) was directly designed in the upstream primers, and the components were connected through multiple rounds of PCR.
  • the Hind III restriction site and the 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 guide, and introduced at the 3'end of the fragment through the downstream guide
  • the restriction site BamHI and the overlapping sequence downstream of the BamHI restriction site corresponding to the Lenti vector The Lenti plasmid is digested with Hind III and BamH I.
  • the inserts with overlapping sequences were ligated to the digested Lenti vector by GIBSON assembly.
  • HEK-293T cells Inoculate HEK-293T cells into a 6-well plate and culture overnight. Before plasmid transfection, ensure that the number of cells per well is about 2*10 7 /ml; before transfection, replace the cells with serum-free DMEM medium; 300 ⁇ l serum-free DMEM was added to 1.5 ⁇ g Lenti recombinant plasmid, 0.75 ⁇ g pMDL plasmid, 0.45 ⁇ g pVSV-G plasmid, 0.3 ⁇ g pREV (mass ratio 5:3:2:1), after slowly blowing evenly, add 9 ⁇ l (1: 3) X-tremeGENE transfection reagent, slowly blow well, stand at room temperature for 15 minutes, and add dropwise to the cell supernatant. After 8 hours, change to DMEM containing 10% FBS to continue the culture; after 60 hours, collect the culture supernatant for infection.
  • the cells were passaged at a ratio of 1/3, and puromycin was added at a concentration of 2.5 ⁇ g/ml for resistance screening; the positive cells were cloned to obtain HEK-293T-Hitone-GFP ⁇ 11 Cloning cell lines.
  • the results of flow cytometry analysis are shown in Figure 5.
  • the results show that the average fluorescence intensity of cells is increased after adding the pH-sensitive peptide INF7 on the basis of TAT, which proves that the pH-sensitive peptide has the effect of breaking the membrane of endocytic vesicles;
  • the cell fluorescence intensity further increased, and the effect of CTSL restriction restriction site N and Furin restriction restriction site Ne was the most significant.
  • the fluorescence microscope observation is shown in Figure 6, and the results show that combining the above two restriction sites (TINNe-GFP ⁇ 1-10) can further significantly improve the escape efficiency of endocytic vesicles.
  • a mutation is introduced at a key site in the N or Ne restriction site to obtain a delivery system-cargo molecule complex containing the mutation, wherein 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 compare the transfection efficiency of the mutant delivery system-cargo molecule complex (Mut) and the delivery system without mutation-cargo molecule complex (WT).
  • Mot mutant delivery system-cargo molecule complex
  • WT delivery system without mutation-cargo molecule complex
  • the total amount of intracellular protein detected was comparable among cells in each group, indicating that pH-sensitive peptides and restriction sites did not increase Cargo's endocytosis efficiency;
  • the protein at the restriction site (TIN-, TINe-, TINNe-) has been digested at 30 minutes, and the amount of protein after digestion does not increase after 3 hours.
  • the single restriction site protein is about 40% Restriction enzyme cleavage occurred, and about 70% of the double restriction site protein was digested; after that, the protein without restriction site (T-, TI-) and the intact protein whose restriction site was not digested were directed to dissolution.
  • Example 2 Application of delivery system in transduction of zinc finger protein ZFP
  • the construction method is as follows: First, the nucleic acid sequence encoding the TAT, INF7, protease cleavage site, and cargo molecule ZFP9 in the delivery system is obtained by PCR amplification, and the parts are connected by multiple rounds of PCR, and in the last round of PCR In the process, the NdeI restriction site and the overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+) were introduced at the 5'end of the fragment through the upstream guide, and the BamHI enzyme was introduced at the 3'end of the fragment through the downstream guide. The cutting site and its overlapping sequence downstream of the BamHI restriction site corresponding to pET-21b(+). The pET-21b(+) plasmid was digested with NdeI and BamHI. The insert with overlapping sequence was ligated to the digestion vector pET-21b(+) by GIBSON assembly.
  • the pTT5 plasmid was digested with Hind III and BamH I.
  • the insert fragments with overlapping sequences were ligated to the pTT5 vector after digestion by GIBSON assembly to obtain pTT5-BFP-6BS plasmid.
  • HEK293T cells Inoculate HEK293T cells into a 12-well plate and culture overnight. Before protein treatment, ensure that the number of cells in each well is about 5*10 6 /ml; the delivery system-ZFP9 complex obtained in 100 ⁇ L/5 ⁇ M 2.2 (ZFP9, T -ZFP9, TI-ZFP9, TINNe-ZFP9) and 5 ⁇ g of pTT5-BFP-6BS plasmid obtained in 2.3 and incubated at 37°C for 30min to fully form a complex, and X-tremeGENE transfection reagent (Roche) was used as a positive control (5 ⁇ g) Plasmid was mixed with 15 ⁇ l transfection reagent, transfected cells under serum-free condition, and replaced with 10% FBS DMEM after 8h to continue culturing); Wash the cells with serum-free DMEM medium three times, then add the complex and incubate for 3h with After washing three times with heparin solution to remove the protein adsorbed on the
  • the results of streaming detection are shown in Figure 15-16.
  • the plasmid bound to ZFP9 can only complete the transcription process when it enters the nucleus. Therefore, only more ZFP9 escapes from the endocytic vesicles can carry the bound plasmid into the nucleus, start the transcription process, and then express blue fluorescent protein.
  • TI-ZFP9 pH-sensitive peptide INF7
  • TI-ZFP9 pH-sensitive peptide INF7
  • TI-ZFP9 pH-sensitive peptide INF7
  • TINNE-ZFP9 protease cleavage site
  • TNF- ⁇ binds to cell surface receptors to induce phosphorylation of RIP3 and form necrosomes of multi-protein complexes.
  • Phosphorylated RIP3 in the necrosomes recruits and phosphorylates Mlkl and the cells enter the necrosis program.
  • the intracellular protein phosphatase 1B (Ppm1b) can inhibit programmed cell necrosis (Necroptosis) by dephosphorylating RIP3.
  • Ppm1b protein has shown great potential in the treatment of the above-mentioned diseases related to programmed cell necrosis .
  • a recombinant protein expression vector containing TAT, INF7, protease cleavage site, and cargo molecule Ppm1b was constructed.
  • the structure diagram of each recombinant protein is shown in Figure 17, and the amino acid sequence of each component is shown in Table 6 below.
  • the construction method is as follows: the nucleic acid sequences of TAT, INF7, protease cleavage site, and Ppm1b are obtained through PCR amplification, and the various parts are connected through multiple rounds of PCR, and in the last round of PCR, the 5 of the fragment is introduced by upstream
  • The'end introduces the NdeI restriction site and the overlapping sequence upstream of the NdeI restriction site corresponding to pET-21b(+), and introduces the BamHI restriction site and its connection with pET-21b at the 3'end of the fragment through the downstream guide (+) The corresponding overlapping sequence downstream of the BamHI restriction site.
  • the pET-21b(+) plasmid was digested with NdeI and BamHI.
  • the inserts with overlapping sequences were ligated to the digestion vector pET-21b(+) by GIBSON assembly.
  • the delivery system proteins Ppm1b, T-Ppm1b, TI-Ppm1b, TINNe-Ppm1b
  • lentivirus to transduce Ppm1b (Lenti-Ppm1b) and lentivirus (Lenti-vec) as controls, package the collected lentivirus with Ppm1b expression sequence and control lentivirus on HEK-293T cells, and after infecting L929 cells for 24 hours, In order to make Ppm1b fully expressed in the cell, the infected L929 cells were re-plated for TNF- ⁇ stimulation.
  • the CRISPR/Cas9 gene editing system uses sgRNA to bind Cas9 protein, sgRNA specifically recognizes the target site, Cas9 binds and cuts DNA double-stranded molecules, and achieves targeted genes through non-homologous end recombination or homologous end repair Edit.
  • Cas9 must enter the nucleus to complete its function. Based on this, we fused the delivery system to express the Cas9 protein to achieve gene editing on eukaryotic cells.
  • the pET-21b(+) plasmid was digested with NdeI and BamHI. The insert with overlapping sequence was ligated to the digestion vector pET-21b(+) by GIBSON assembly.
  • Nickel column initial purity Transform the expression plasmid described in 4.1 into the expression strain E.coli BL21(DE3); select a single colony from the transformed plate and inoculate it in 5ml LB liquid medium containing ampicillin resistance and cultivate overnight, then Transfer 1ml of the overnight cultured bacterial solution to 500ml LB liquid medium containing ampicillin resistance, culture at 37°C and 180rpm until the bacterial solution OD 600 is around 0.6, and then add the inducer IPTG to a final concentration of 0.2mM, 25°C Induction for 8h; after the induction of expression, collect the bacteria after centrifugation at 7000g for 10 minutes at 4°C; then use 10ml protein purification equilibration buffer (5% glycerol, 30mM TB8.0, 50mM glycerol, 500mM sodium chloride, 25mM glucose ) Resuspend the bacterial cells and ultrasonically disrupt them.
  • 10ml protein purification equilibration buffer 5% glycerol
  • protein purification elution buffer 5% glycerol 30mM TB8.0, 50mM glycerol, 500mM sodium chloride, 25mM glucose, 250mM imidazole
  • Cation exchange column purification Dialysis the target protein collected from the initial purity of the nickel column into an equilibrium buffer (30mM TB8.0, 50mM glycerol, 25 0mM sodium chloride, 25mM glucose, adjusted to pH 7.2), and load the sample To the Sulphopropyl (SP) cation exchange column of AKTA protein purification system; then use equilibration buffer and high salt eluent (30mM TB8.0, 50mM glycerol, 2M sodium chloride, 25mM glucose, adjust pH to 7.2) Gradient elution was performed in different proportions to obtain the target protein.
  • the protein concentration can be determined according to a spectrophotometer or a BCA protein concentration determination kit. Each purified fusion protein is aliquoted and stored at -20°C. The SDS-PAGE results of each protein are shown in Figure 21.
  • Cas9 and sgRNA After Cas9 and sgRNA are combined, they can specifically recognize the target site, and non-homologous end recombination will occur if the donor is not provided. Therefore, the sgRNA recognition site and the two red fluorescent protein genes that are not in the reading frame (the difference between dsRed and mCherry) are integrated into the genome of HEK-293T cells by means of lentivirus infection. If Cas9 causes the sgRNA recognition site When the DNA sequence on the dot is recombined with homologous ends, it will cause the red fluorescent protein gene that is not in the reading frame to enter the reading frame and be expressed, and the cell changes from no fluorescence to red fluorescence. The number of fluorescent cells can be used to evaluate the efficiency of the delivery system to transduce the genetically engineered enzyme Cas9 ( Figure 22).
  • the coding sequence of dsRed (SEQ ID NO: 35) and mCherry (SEQ ID NO: 36) were obtained by PCR amplification.
  • the recognition site DNA sequence of sgRNA (SEQ ID NO: 37) was directly designed in the primers.
  • the components are connected, and the Hind III restriction site and the overlapping sequence upstream of the Hind III restriction site corresponding to the Lenti vector are introduced at the 5'end of the fragment through the upstream guide during the last round of PCR.
  • the BamHI restriction site and the 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 guide.
  • the Lenti plasmid is digested with Hind III and BamH I.
  • the inserted fragments with overlapping sequences were ligated to the digested Lenti vector by GIBSON assembly to construct a successful plasmid map ( Figure 23).
  • HEK293T cells Inoculate HEK293T cells into a 6-well plate and culture overnight. Before plasmid transfection, ensure that the number of cells per well is about 2*10 7 /ml; before transfection, replace the cells with serum-free DMEM medium; in 300 ⁇ l Serum-free DMEM was added with 1.5 ⁇ g Lenti recombinant plasmid (RFP reporter), 0.75 ⁇ g pMDL plasmid, 0.45 ⁇ g pVSV-G plasmid, 0.3 ⁇ g pREV plasmid (the mass ratio is 5:3:2:1), slowly blow well and let stand After 5 minutes, add 9 ⁇ l of X-tremeGENE Transfection Reagent, slowly blow evenly, after standing at room temperature for 15 minutes, add to the cell supernatant, 8h later, change to DMEM containing 10% FBS to continue the culture; 60h later, collect the culture supernatant, 4 Store at °C.
  • RFP reporter 1.5 ⁇ g L
  • the cells were passaged at a ratio of 1/3, and puromycin was added at a concentration of 2.5 ⁇ g/ml for resistance screening; the positive cells obtained from the screening were cloned to obtain the HEK293T-RFPreporter monoclonal cell line .
  • gRNA adopts the method of transfection of transcription plasmid and transcribed into gRNA in the cell.
  • the DNA sequence corresponding to gRNA is produced by primer annealing and overlapping.
  • primer design the sticky end of the AflII restriction site is directly introduced;
  • gRNA -Cloning vector is treated with AflII single enzyme digestion; vector and insert are ligated by T4DNA ligase using their respective sticky ends.
  • Transfection of gRNA transcription plasmid Inoculate HEK-293T-RFP reporter cell line into a 12-well plate and culture overnight. Before transfection, ensure that the number of cells per well is about 2.5*10 6 /ml; before transfection, replace the cells with Serum-free DMEM; add 1 ⁇ g gRNA-GM3 transcription plasmid to 100 ⁇ l of serum-free DMEM, slowly blow well, let stand for 5 minutes, add 3 ⁇ l X-tremeGENE transfection reagent, blow well and let stand for 15 minutes, then add to the cell supernatant, After 8 hours, it was replaced with 10% FBS DMEM.
  • Delivery system transduction of Cas9 12h after gRNA transcription plasmid transfection (4h after changing to DMEM with serum), rinse the cells with serum-free DMEM three times, and add 5 ⁇ M of the delivery system in 4.2 under the environment of serum-free DMEM- Incubate the Cas9 complex for 3h; change to 10% FBS DMEM to continue the culture, observe and flow cytometric analysis of red fluorescent protein expression at 48h.
  • CTSL and Furin specific sites N and Ne significantly promotes the escape process of the carried Cas9, and more Cas9 enters under the action of the nuclear signal
  • the nucleus after binding to the GM3sgRNA formed by intracellular transcription, specifically recognizes the target sequence, and homologous end repair occurs near this site, so that the red fluorescent gene enters the reading frame and is expressed.
  • Example 5 Establishment of delivery system based on non-covalent connection
  • connection mode of the fusion protein and cargo of the present invention can also be non-covalently connected through protein domains, which we call Adapter pairs, such as heterodimers with strong interaction.
  • Acid zipper Figure 25.
  • Two anti-parallel leucine zipper domains can spontaneously combine to form oligomers due to their chimeric space characteristics and charge arrangement.
  • NZ and CZ are such a pair of leucine zippers that can be combined with each other.
  • the HEK293T cells stably expressing Histone- ⁇ 11 are transduced, and the escape efficiency of endocytic vesicles can be evaluated by the ratio of GFP and the relative fluorescence intensity.
  • the feasibility of NZ-CZ adapter to connect the fusion protein to the cargo In order to evaluate the feasibility of NZ-CZ adapter to connect the fusion protein to the cargo.
  • TrxA is added as a solubilizing label.
  • the structure diagram of the recombinant protein is shown in Figure 26, and the amino acid sequence of the delivery system is shown in the following table.
  • the construction method is as follows: First, using the aforementioned vector as a template, the TAT, INF7, N, Ne, and NZ sequences encoding the delivery system, as well as the CZ sequence, and the nucleic acid sequence of the cargo molecule sGFP ⁇ 1-10 are obtained by PCR amplification For TrxA-TINNe-NZ, in the last round of PCR, the BamHI restriction site and the upstream overlapping sequence of the BamHI restriction site corresponding to pET-32a(+) were introduced at the 5'end of the fragment through the upstream primer , Introduce the HIindIII restriction site and the overlapping sequence downstream of the HIindIII restriction site corresponding to pET-32a(+) at the 3'end of the fragment through the downstream guide.
  • the pET-32a(+) plasmid was digested with BamHI and HIindIII. Connect the insert with overlapping sequence to the digestion vector pET-32a(+) by GIBSON assembly; for CZ-GFP ⁇ 1-10, in the last round of PCR, the NdeI enzyme was introduced at the 5'end of the fragment through the upstream guide The cutting site and the overlapping sequence upstream of the NdeI restriction site corresponding to pET21b(+), introduce the BamHI restriction site and the BamHI enzyme corresponding to pET-21b(+) at the 3'end of the fragment through the downstream guide Overlapping sequences downstream of the cut site.
  • the pET-21b(+) plasmid was digested with NdeI and BamHI. The insert with overlapping sequence was ligated to the digestion vector pET-21b(+) by GIBSON assembly.
  • the MFI value of the TrxA-TINNe-NZ+CZ-GFP ⁇ 1 ⁇ 10 group is close to 20, which is significantly higher than that of the control group, indicating that under the action of NZ and CZ, TrxA-TINNe and GFP ⁇ 1 ⁇ 10 have a certain degree of binding, and pass The effect of TINNe makes GFP ⁇ 1 ⁇ 10 enter the cell and escape.
  • the fluorescence intensity of the adapter connection method is still lower than the TINNe-GFP ⁇ 1 ⁇ 10 of the fusion expression connection method, this experiment proves that the adapter based on NZ-CZ or similar can be used as the connection method between the delivery system and the goods, and can effectively deliver the goods. Enter the cell and escape from the vesicle.

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Abstract

一种融合蛋白及含有所述融合蛋白的复合物，其可用于胞内递送货物分子。所述融合蛋白及复合物能够实现货物分子从内吞小泡的高效释放，显著提升货物分子的细胞质递送效率。一旦在细胞质中可获得货物分子时，它们就可发挥与其相关的任何作用。所述融合蛋白及复合物提供了用于影响细胞的生物学机制和途径的有效手段，其可用于研究、治疗、诊断等诸多领域。

Description

用于胞内递送分子的复合物 技术领域
本发明涉及分子生物学领域，更具体的说，涉及用于胞内递送货物分子的融合蛋白及复合物。
背景技术
细胞膜作为选择性透过性屏障对细胞存活和功能至关重要，虽然小分子物质可以通过细胞的天然过程或脂质双层的直接扩散穿过细胞膜，但是在大多数情况下，外源性的活性生物大分子等细胞内载物从细胞质膜的有效通过，仍然是细胞运送过程的主要障碍。因此，一种能够有效提高细胞内载物向活细胞运输效率的分子转运工具对于其在生物医药等领域的应用极为重要。
细胞穿膜肽(Cell-penetrating peptides,CPPs)是目前用于实现细胞摄取过程的最普遍和有效的载体之一。细胞穿膜肽，通常含有5-30个氨基酸，可以以化学交联、融合表达或非共价结合的方式携带生物大分子穿过细胞膜进入胞内。迄今，已有上百种来源于天然蛋白或人工合成的CPPs被报道并用于细胞内载物的胞内递送研究，根据其化学性质的不同，可以分为三类:(1)阳离子型，富含精氨酸和赖氨酸残基，在生理pH下具有强正电荷；(2)两亲型，包含极性和非极性区域，除了分布在整个序列中的赖氨酸和精氨酸外，它们还富含疏水残基，如缬氨酸、亮氨酸、异亮氨酸和丙氨酸等；(3)疏水型，主要含有非极性氨基酸。CPPs通过其强正电荷或疏水基团与细胞膜表面负电荷或疏水脂双层相互作用，当携带小分子时可以采用非耗能的方式直接转位穿过细胞膜，当携带生物大分子时基本上采用能量依赖的胞吞作用(endocytosis)进入胞内。由于CPPs在介导入胞时用量低、转运所需时间段、剂量可控、操作简单以及毒副作用低等优点，目前CPP已在体外及体内被广泛应用于转运多肽、蛋白、寡核苷酸、质粒、脂质体和金属离子，小分子荧光，纳米颗粒等等。
尽管CPPs在转导生物大分子上具有诸多优势，但仍存在一些局限性，其中首先需要解决的是其较低的跨膜递送效率。目前普遍认为，在转导内载物入胞时，CPPs与细胞膜表面相互作用，然后通过细胞内吞(endocytosis)进入胞内。细胞内吞可根据其内吞机制的不同可分为四种主要方式:巨胞饮作用(macropinocytosis)、网格蛋白介导的内吞(clathrin-mediated endocytosis)、胞膜窖/脂筏介导的内吞(caveolae/lipid raft-mediated  endocytosis)以及不依赖于网格蛋白/胞膜窖的内吞(clathrin/caveolae-independent endocytosis)等。这些不同的细胞内吞方式最终都会生成内吞小泡(endosome)，并由早期内吞小泡(early endosome)向晚期内吞小泡(late endosome)单向成熟并伴随小泡内pH逐步降低，最终与溶酶体(lysosome)融合。细胞内载物需要在进入溶酶体之前从内吞小泡逃逸(endosomal escape)进入胞浆，否则，将最终进入溶酶体并被降解而不能发挥功能。已有研究表明，CPPs携带的生物大分子仅有1％能在内吞小泡逃逸成功，大部分最终导向溶酶体而被降解。因此，细胞内载物的小泡逃逸是CPPs胞内递送过程的关键限制因素，其效率决定了总体递送效率。
对于提升小泡逃逸效率，主要策略为在成熟酸化的过程中破坏小泡囊膜的完整性，从而使其内容物(包括被递送的细胞内载物)释放到胞浆中。有报道可以通过在体系中加入缓冲剂，比如氯喹(chloroquine)，甲胺(methylamine)，或者氯化铵(ammonium chloride)，以物理性方式促进内吞小泡的渗透破裂，但因其较强的细胞毒性阻碍了在临床上的应用。而目前最有效的方式是利用来源于病毒、细菌、动植物或者人类的pH敏感肽(pH sensitive fusogenic peptides)。pH敏感肽含有一定比例的疏水氨基酸，且在低pH值的情况下会发生构象的剧烈改变。在CPPs介导细胞内吞后，内吞小泡在成熟酸化的过程中，一旦pH值下降到临界点，与CPPs偶联的pH敏感肽发生构象改变并与小泡囊膜的脂双层结合从而剧烈扰动磷脂双层膜的完整性，在其上形成小孔或导致囊膜裂解，最后将被转运的生物大分子释放进入胞浆。目前较为常用的pH敏感肽为流感病毒血凝素抗原茎部区的一段多肽(以下简称HA2)，以及基于HA2人工改造后毒性更低、破膜效果更好的INF7，二者与CPPs及蛋白分子融合表达后，能够提升转运蛋白分子的内吞小泡逃逸效率。然而，虽然融合pH敏感肽后的CPPs大分子胞内递送系统的内吞小泡逃逸效率确实得到提高，但相当一部分的被转运大分子仍留存在内吞小泡内(以荧光蛋白作为被转运大分子可见明显的点状分布)。
因此，仍然需要开发新的递送系统，以实现被转运的生物大分子从内吞小泡的高效释放。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料地发现将pH敏感肽与特定蛋白酶识别序列联用作为递送载体能够显著提高货物分子从内吞小泡的释放，从而显著提升货物分子的细胞质递送效率，使货物分子充分发挥其相应的生物学功能。基于这些发现， 本发明人开发了能够实现高效细胞质递送的载体系统。
融合蛋白
因此，本发明第一方面提供了一种融合蛋白，其包含细胞穿膜肽、pH敏感融合肽(pH-sensitive fusogenic peptide)以及蛋白酶识别序列，其中，所述蛋白酶选自弗林蛋白酶和/或溶酶体半胱氨酸蛋白酶。
在某些实施方案中，所述弗林蛋白酶识别序列包含下述序列或由其组成：R-X 1-X 2-R ↓(SEQ ID NO:1)，其中，X 1为任意氨基酸，X 2为K或R，↓表示切割位点。
在某些实施方案中，所述弗林蛋白酶识别序列包含下述序列或由其组成：R-R-X 1-X 2-R ↓(SEQ ID NO:2)。
在某些实施方案中，X 1选自丙氨酸(A)、精氨酸(R)、天冬氨酸(D)、半胱氨酸(C)、谷氨酰胺(Q)、谷氨酸(E)、组氨酸(H)、异亮氨酸(I)、甘氨酸(G)、天冬氨酸(N)、亮氨酸(L)、赖氨酸(K)、甲硫氨酸(M)、苯丙氨酸(F)、辅氨酸(P)、丝氨酸(S)、苏氨酸(T)、色氨酸(W)、酪氨酸(Y)和缬氨酸(V)。
在某些实施方案中，所述弗林蛋白酶识别序列包含下述序列或由其组成：RRHKR ↓(SEQ ID NO:3)。
在某些实施方案中，所述弗林蛋白酶识别序列包含下述序列或由其组成：QSVASSRRHKR ↓FAGV(SEQ ID NO:4)。
在某些实施方案中，所述溶酶体半胱氨酸蛋白酶选自组织蛋白酶B、组织蛋白酶C、组织蛋白酶X、组织蛋白酶S、组织蛋白酶L、组织蛋白酶D或组织蛋白酶H。
在某些实施方案中，所述溶酶体半胱氨酸蛋白酶是组织蛋白酶L。
在某些实施方案中，所述组织蛋白酶L识别序列包含下述序列或由其组成：NNTHDLVGDVRLAGV(SEQ ID NO:6)。
在某些实施方案中，所述蛋白酶识别序列包含弗林蛋白酶识别序列和组织蛋白酶L识别序列。在某些实施方案中，所述蛋白酶识别序列是单链多肽，其从N端至C端包含弗林蛋白酶识别序列和组织蛋白酶L识别序列，或者其从N端至C端包含组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在某些实施方案中，所述蛋白酶识别序列包含RRHKR(SEQ ID NO:3)和NNTHDLVGDVRLAGV(SEQ ID NO:6)。在某些实施方案中，所述蛋白酶识别序列包含SEQ ID NO:4和SEQ ID NO:6。
在本发明中，术语“pH敏感融合肽(pH-sensitive fusogenic peptide)”与“pH敏感肽”可互换使用，其是指一类能够在酸性条件(例如，pH<6.5)下发生构象改变从而促进与内吞小泡囊膜融合的多肽。当pH敏感肽被细胞内吞后，内吞小泡在成熟酸化的过程中，一旦pH值下降到临界点，此类多肽发生构象改变并与小泡囊膜的脂双层结合从而剧烈扰动磷脂双层膜的完整性，在其上形成小孔或导致囊膜裂解，从而将被转运的生物大分子释放进入胞浆。这类多肽是本领域熟知的，并描述于例如，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，其全部通过引用并入本文。
能够用于本发明的融合蛋白的pH敏感肽可以选自下列的蛋白或多肽，或者源自选自下列蛋白或多肽：
病毒蛋白来源：HA2(流感病毒)及其突变体KALA,GALA；五邻体蛋白(penton base)(腺病毒或鼻病毒)，gp41(HIV)，L2(乳头瘤病毒)，包膜蛋白(西尼罗河病毒(West Nile virus))；
细菌蛋白来源：单增李斯特氏菌溶血素O(Listeriolysin O(LLO))，肺炎球菌溶血素(Pneumococcal pneumolysin(PLO))，链球菌溶血素O(Streptococcal streptolysin O(SLO))，白喉毒素(Diphtheria toxin)，铜绿假单胞菌外毒素A(Pseudomonas aeruginosa exotoxin A)，志贺毒素(Shiga toxin)，霍乱毒素(Cholera toxin)；
植物蛋白来源：蓖麻毒素(Ricin)，皂草素(Saporin)，Gelonin毒素；
人类/动物蛋白来源：人降钙素，成纤维细胞生长因子受体，蜂毒素(Melittin)；
人工合成多肽：(R-Ahx-R)(4)AhxB，穿透素(Penetratin(pAntp))，EB1，牛朊蛋白(Bovine prion protein(bPrPp))，甜箭肽(Sweet Arrow Peptide(SAP)),聚L-组氨酸(Poly(L-histidine))，脯氨酸富含肽(Proline-rich)。
在某些实施方案中，所述pH敏感融合肽选自流感病毒HA2(SEQ ID NO:38)或其突变体、蜂毒素(Melittin)(SEQ ID NO:41)，及其任意组合。在某些实施方案中，所述流感病毒HA2的突变体选自INF7(SEQ ID NO:8)、KALA(SEQ ID NO:39)或GALA(SEQ ID NO:40)。
在某些实施方案中，所述pH敏感融合肽包含INF7。在某些实施方案中，所述pH敏感融合肽包含下述序列或由其组成：SEQ ID NO:8。
在本发明中，术语“细胞穿膜肽(cell penetrating peptide，CPP)”又称为“细胞穿透肽”、“蛋白质转导域(protein translocation domain，PTD)、“Trojan horse peptides”或“转导肽(transduction peptide)”等，其是指，能够促进各种分子(例如，各种大分子包括蛋白或核酸)的细胞摄取的多肽。这类多肽是本领域熟知的，并描述于例如，Stewart KM,et al.Org Biomol Chem.2008Jul 7；6(13):2242-55以及中国专利申请CN101490081A(其全部通过引用并入本文)；或者可以通过本领域已知的方法获得，例如详细描述于美国专利申请US 2008/0234183中的方法，其全部通过引用并入本文。
能够用于本发明的融合蛋白的CPP包括但不限于：阳离子型:Penetratin、HIV-TAT-47-57、HIV-1Rev 34–50、FHV coat-35-49、低聚精氨酸(Oligoarginines)(R9–R12)、CCMV Gag-7-25、S413-PV、VP22、BP16、DPV3、DPV6、FAH外壳蛋白、鱼精蛋白1(Protamine 1)、人cJun、Engrailed-2、Islet-1、HoxA-13、TP10等；两亲性型：转运肽(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肽(19–40)、Buforin 2、Crotamine、Azurin p18、hCT肽(18-32)、S413-PVrev等；疏水型：Kaposi's肉瘤成纤维细胞生长因子、源自Caiiman crocodylus的Ig轻链的信号肽、整合素β3片段、Grb2-SH2结构域、HIV-1gp41(1-23)、HBV移位基序(translocation motif)、精卵融合蛋白(89–111)、人降钙素(9–32)、Pep-7、C105Y、K-FGF等。
此外，用作本发明的融合蛋白中的CPP还可以选自与如上所述的任何多肽序列具有大约60、70、80、90、95、99％或100％的序列同一性的多肽序列，只要该多肽序列仍然保留其生物学活性，即，促进分子的细胞摄取。
在某些实施方案中，所述细胞穿膜肽选自穿透素(Penetratin)(SEQ ID NO:42)、Tat衍生肽、Rev(34-50)(SEQ ID NO:44)、VP22(SEQ ID NO:45)、转运肽(transportan)(SEQ ID NO:46)、Pep-1(SEQ ID NO:47)、Pep-7(SEQ ID NO:48)，及其任意组合。在某些实施方案中，所述Tat衍生肽选自Tat(48-60)(SEQ ID NO:10)或Tat(47-57)(SEQ ID NO:43)。
在某些实施方案中，所述细胞穿膜肽包含Tat衍生肽，例如Tat(48-60)。在某些实施方案中，所述细胞穿膜肽包含下述序列或由其组成：SEQ ID NO:10。
在一些实施方案中，本发明的融合蛋白从N端至C端包含所述pH敏感融合肽、细胞穿膜肽和蛋白酶识别序列。在某些实施方案中，所述融合蛋白从N端至C端包含所述pH敏感融合肽、细胞穿膜肽、弗林蛋白酶识别序列和组织蛋白酶L识别序列。在某些实施方案中，所述融合蛋白从N端至C端包含所述pH敏感融合肽、细胞穿膜肽、组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在另一些实施方案中，本发明的融合蛋白从N端至C端包含所述细胞穿膜肽、pH敏感融合肽和蛋白酶识别序列。在某些实施方案中，所述融合蛋白从N端至C端包含所述细胞穿膜肽、pH敏感融合肽、弗林蛋白酶识别序列和组织蛋白酶L识别序列。在某些实施方案中，所述融合蛋白从N端至C端包含所述细胞穿膜肽、pH敏感融合肽、组织蛋白酶L识别序列和弗林蛋白酶识别序列。
在某些实例性实施方案中，所述融合蛋白包含选自下列的序列或由其组成：SEQ ID NOs:12-14任一项所示的序列。
某些实施方案中，所述融合蛋白在其N端可进一步包含蛋白标签。在某些实施方案中，所述蛋白标签具有促溶作用。在某些实施方案中，所述蛋白标签选自TrxA、SUMO、NusA、MBP、GST。
本发明第二方面还提供了一种融合蛋白，其在第一方面所述的融合蛋白的基础上进一步包含特异性结合序列，所述特异性结合序列允许另外的分子(例如多肽、蛋白或核酸(如DNA))与其发生特异性结合。在本文中，术语“特异性结合”是指两分子间的非随机的结合反应，如抗体(或其抗原结合片段)和其所针对的抗原(或抗原表位)之间的反应，或者两个氨基酸序列(例如反向平行的两个亮氨酸拉链结构域)之间形成异源二聚体的反应。
在某些实施方案中，所述特异性结合序列包含亮氨酸拉链，并且所述亮氨酸拉链能够与其反向序列形成异源二聚体。在某些实施方案中，所述特异性结合序列包含亮氨酸拉链NZ或CZ。本领域已知，亮氨酸拉链NZ与CZ互为反向序列，两者之间具有较强相互作用力，从而反向平行的亮氨酸拉链NZ与CZ可形成异源二聚体。在某些实施方案中，所述亮氨酸拉链NZ包含SEQ ID NO:49所示的序列。在某些实施方案中，所述亮氨酸拉链CZ包含SEQ ID NO:50所示的序列。
在一些实施方案中，所述特异性结合序列包含SEQ ID NO:49所示的序列。在另一 些实施方案中，所述特异性结合序列包含SEQ ID NO:50所示的序列。
在某些实施方案中，所述特异性结合序列位于所述蛋白酶识别序列的C端。在某些实施方案中，所述融合蛋白在其C端包含所述特异性结合序列。
某些实施方案中，所述融合蛋白在其N端可进一步包含蛋白标签。在某些实施方案中，所述蛋白标签具有促溶作用。在某些实施方案中，所述蛋白标签选自TrxA、SUMO、NusA、MBP、GST。
融合蛋白的制备
本发明的融合蛋白可以本领域已知的各种方法来制备，例如，通过基因工程方法(重组技术)产生，也可以通过化学合成方法(例如Fmoc固相方法)产生。本发明的融合蛋白不受其产生方式的限定。
因此，在另一方面，本发明提供了一种分离的核酸分子，其包含编码本发明第一或第二方面的融合蛋白的核苷酸序列。
在另一方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，所述载体是例如质粒，粘粒，噬菌体等。
在另一方面，本发明提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一方面，提供了制备本发明第一或第二方面的融合蛋白的方法，其包括，在允许所述融合蛋白表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述融合蛋白。
复合物
在另一方面，本发明提供了一种复合物，其包含本发明第一或第二方面的融合蛋白以及货物分子(cargo)。所述货物分子可以是任意的分子。
在某些实施方案中，所述货物分子选自核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物。
在某些实施方案中，所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合。
在某些实施方案中，所述货物分子的分子量小于10000Da，例如小于5000Da，小于 3000Da，或小于1000Da。
在某些实施方案中，所述货物分子包含可检测的标记，例如酶、放射性核素、荧光染料、化学发光物质或生物素等。
在某些实施方案中，所述货物分子包含表位标签(epitope tag)、报告基因序列和/或核定位信号(NLS)序列。在某些实施方案中，所述货物分子是肽或蛋白。
可用于货物分子中的表位标签是本领域技术人员熟知的，其实例包括但不限于His、V5、FLAG、HA、Myc、VSV-G、Trx等，并且本领域技术人员已知如何根据期望目的(例如，纯化、检测或示踪)选择合适的表位标签。在某些实例性实施方案中，所述货物分子包含His标签。
可用于货物分子中的报告基因序列是本领域技术人员熟知的，其实例包括但不限于GST、HRP、CAT、GFP、HcRed、DsRed、CFP、YFP、BFP等。
可用于货物分子中的核定位信号(NLS)序列是本领域技术人员熟知的，其实例包括但不限于SV40病毒大T抗原的NLS。在某些实例性实施方案中，所述NLS序列如SEQ ID NO:15所示。
在一些实施方案中，本发明的融合蛋白与货物分子是融合的，其中所述货物分子是肽或蛋白质。在某些实施方案中，所述融合蛋白如第一方面定义。
在某些实施方案中，所述货物分子融合至所述融合蛋白的N端或C端。在某些实施方案中，所述货物分子融合至所述融合蛋白的C端。
在一些实施方案中，本发明的复合物包含单链多肽，其从N端至C端包含：所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列和货物分子。在某些实施方案中，所述单链多肽从N端至C端包含所述pH敏感融合肽、细胞穿膜肽、弗林蛋白酶识别序列、组织蛋白酶L识别序列和货物分子。在某些实施方案中，所述单链多肽从N端至C端包含所述pH敏感融合肽、细胞穿膜肽、组织蛋白酶L识别序列、弗林蛋白酶识别序列和货物分子。
在另一些实施方案中，本发明的复合物包含单链多肽，其从N端至C端包含：所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列和货物分子。在某些实施方案中，所述单链多肽从N端至C端包含所述细胞穿膜肽、pH敏感融合肽、弗林蛋白酶识别序列、组织蛋白酶L识别序列和货物分子。在某些实施方案中，所述单链多肽从N端至C端包含所述细胞穿膜肽、pH敏感融合肽、组织蛋白酶L识别序列、弗林蛋白酶识别序列和货物分子。
在某些实例性实施方案中，所述货物分子是锌指蛋白(例如ZFP9)、蛋白磷酸酶(例如Ppm1b)或Cas效应蛋白(例如Cas9)。表述“Cas效应蛋白”是指CRISPR-Cas系统的效应蛋白。在某些实例性实施方案中，所述锌指蛋白或Cas效应蛋白包含NLS序列。
在另一些实施方案中，本发明的融合蛋白与货物分子化学偶联(chemical coupling)。所述“化学偶联”指在融合蛋白所包含的反应基团与货物分子所包含的反应基团之间的化学反应中获得的键合，所述化学反应后两个部分通过共价键连接。在上述化学反应(偶联反应)之前，可以用接头分子在独立的反应中对融合蛋白、货物分子或二者进行修饰以使它们分别包含该化学偶联所需的反应基团。用于修饰融合蛋白或货物分子的接头分子的选择取决于所使用的偶联策略。在某些实施方案中，所述融合蛋白如第一方面定义。
在某些实施方案中，所述共价键为二硫键、磷酸二酯键、硫代磷酸酯键、酰胺键、胺键、硫醚键、醚键、酯键或碳-碳键。
在某些实施方案中，所述货物分子偶联至所述融合蛋白的N端或C端。在某些实施方案中，所述货物分子偶联至所述融合蛋白的C端。
在某些实施方案中，所述货物分子是核酸。
在另一些实施方案中，本发明的融合蛋白与货物分子非共价连接。
在某些实施方案中，所述融合蛋白与货物分子通过静电作用缀合。在某些实施方案中，所述货物分子是核酸。
在某些实施方案中，所述融合蛋白如第二方面定义，所述融合蛋白通过其所包含的特异性结合序列与货物分子通过非共价连接。在此类实施方案中，所述货物分子包含能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域。
在某些实施方案中，所述能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域是氨基酸序列。
在某些实施方案中，所述货物分子是肽或蛋白质。在某些实施方案中，所述货物分子在其N端包含所述能够与所述融合蛋白中的特异性结合序列发生特异性结合的氨基酸序列。
在某些实施方案中，所述融合蛋白中的特异性结合序列包含亮氨酸拉链，所述货物分子包含所述亮氨酸拉链的反向序列，从而所述亮氨酸拉链能够与该反向序列形成异源 二聚体。
在某些实施方案中，所述融合蛋白中的特异性结合序列包含亮氨酸拉链NZ(例如，SEQ ID NO:49所示的序列)，所述货物分子包含亮氨酸拉链CZ(例如，SEQ ID NO:50所示的序列)。
在某些实施方案中，所述融合蛋白中的特异性结合序列包含亮氨酸拉链CZ(例如，SEQ ID NO:50所示的序列)，所述货物分子包含亮氨酸拉链NZ(例如，SEQ ID NO:49所示的序列)。
复合物的制备
本发明的复合物可以本领域已知的各种方法来制备，例如，通过基因工程方法(重组技术)产生，也可以通过化学合成方法(例如Fmoc固相方法)产生。本发明的复合物不受其产生方式的限定。
在一些实施方案中，当所述复合物包含融合的融合蛋白与货物分子时，本发明的复合物可以通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码该复合物的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的复合物。
因此，在另一方面，本发明提供了一种分离的核酸分子，其包含编码如上所述的复合物的核苷酸序列。
在另一方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含如上所述的分离的核酸分子。在某些实施方案中，所述载体是例如质粒，粘粒，噬菌体等。
在另一方面，本发明提供了一种宿主细胞，其包含如上所述的分离的核酸分子或载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
在另一方面，提供了制备如上所述的复合物的方法，其包括，在允许所述复合物表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述复合物。
在另一些实施方案中，当所述复合物包含化学偶联的融合蛋白与货物分子时，本发明的复合物可以通过下述实例性方法获得：在允许所述融合蛋白和货物分子所分别包含的反应基团发生化学反应的条件下，将所述融合蛋白与货物分子混合，以使得所述两个部分通过共价键连接。在某些实施方案中，所述方法还包括：使用接头分子对融合蛋白、 货物分子或二者进行修饰以使它们分别包含上述化学反应所需的反应基团。在某些实施方案中，所述货物分子是核酸。
在另一些实施方案中，当所述复合物包含通过静电作用缀合的融合蛋白与货物分子时，本发明的复合物可以通过下述实例性方法获得：(1)使本发明的融合蛋白与货物分子混合，以形成混合物；和(2)培育所述混合物以使得所述融合蛋白与货物分子形成复合物。在某些实施方案中，所述货物分子是核酸。
在另一些实施方案中，当所述复合物包含通过特异性结合而非共价连接的融合蛋白与货物分子时，本发明的复合物可以通过下述实例性方法获得：(1)使本发明第二方面所述的融合蛋白与货物分子混合，所述货物分子包含与所述融合蛋白中的特异性结合序列发生特异性结合的结构域；和(2)培育所述混合物以使得所述融合蛋白与货物分子形成复合物。在某些实施方案中，所述货物分子是多肽或蛋白。
组合物
当本发明的融合蛋白能够通过非共价作用与货物分子连接时，通过将融合蛋白与货物分子混合即可获得递送复合物。因此，在另一方面，本发明还提供了一种组合物，其包含本发明的融合蛋白以及货物分子。
在某些实施方案中，所述货物分子选自核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物。在某些实施方案中，所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合。
在某些实施方案中，所述货物分子选自核酸。
在某些实施方案中，所述货物分子是多肽或蛋白。
在某些实施方案中，所述融合蛋白如第二方面定义。所述货物分子包含能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域。
用途及方法
本发明的融合蛋白能够使货物分子从内吞小泡中高效释放，一旦在细胞质中可获得货物分子时，它们就可发挥与其相关的任何作用。因此，本发明的融合蛋白可用作胞内递送试剂，从而进一步用于研究以及治疗和诊断应用。
因此，在另一方面，本发明提供了一种药物组合物，其含有本发明的融合蛋白、复合物、组合物、分离的核酸分子、载体或宿主细胞，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述复合物或组合物所包含的货物分子是药学活性剂。
在某些实施方案中，所述复合物或组合物所包含的货物分子是可检测的标记。所述标记可用于诊断、用于研究药物处置(例如，吸收、分布、代谢、排泄)、用于研究治疗或药物的功效或副作用等。
在另一方面，本发明还涉及本发明的融合蛋白、包含编码所述融合蛋白的核苷酸序列的分离的核酸分子、载体或宿主细胞，作为递送试剂(例如胞内递送试剂和/或转染试剂)用于制备药物的用途。
在另一方面，本发明还涉及本发明的复合物或组合物、或包含编码所述复合物或组合物的核苷酸序列的分离的核酸分子、载体或宿主细胞，在制备用于治疗疾病的药物中的用途；其中，所述复合物或组合物所包含的货物分子能够治疗所述疾病。
在某些实施方案中，所述疾病是与细胞程序性坏死相关的疾病，其中，所述货物分子包含蛋白磷酸酶1B。在某些实施方案中，所述与细胞程序性坏死相关的疾病包括肝损伤(例如药源性肝损伤)、炎性疾病、缺血再灌注损伤和/或神经退行性疾病。
本发明的融合蛋白、复合物或组合物、或药物组合物可以为医学领域已知的任何形式，例如，可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)、吸入剂、喷雾剂等形式。优选剂型取决于预期的给药方式和治疗用途。
本发明的融合蛋白、复合物或组合物、或药物组合物，可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、直肠、肠胃外或局部给药。
一种示例性施用途径是口服给药。用于口服给药的液体剂型包括药学上可接受的乳剂、微乳剂、溶液剂、悬浮剂、糖浆剂、酏剂等。除活性化合物以外，液体剂型可含有本领域常用的惰性稀释剂，例如水或其它溶剂、增溶剂和乳化剂，例如乙醇、异丙醇、醋酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、二甲基甲酰胺、油类(例如棉籽油、花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇和脱水山梨糖醇的脂肪酸酯及其混合物。除惰性稀释剂以外，口服给药的液体剂型也可包括助剂，例如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和芳香剂等。用于 口服给药的固体剂型包括胶囊剂、片剂、丸剂、锭剂、粉剂、颗粒剂等。除活性化合物以外，固体剂型可含有药学上可接受的惰性赋形剂或载体，例如填充剂(如乳糖、蔗糖、葡萄糖、甘露醇、淀粉、微晶纤维素、半乳糖、交联聚维酮和硫酸钙)；粘合剂(如羧甲基纤维素、海藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯胶)；湿润剂(如鲸蜡醇和单硬脂酸甘油酯)；崩解剂(如琼胶、碳酸钙、淀粉、褐藻酸、羧甲基纤维素钠、羧甲基淀粉钠)；润滑剂(如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、月桂基硫酸钠)；及其混合物。
本发明的融合蛋白、复合物或组合物、或药物组合物也可通过非口服途径给药。
因此，另一种示例性的施用途径是肠胃外给药，例如，皮下注射、静脉注射、腹膜内注射、肌肉注射、胸骨内注射和注入。用于肠道外给药的剂型可以为注射制剂，包括注射液、注射用无菌粉末或注射用浓溶液。除活性化合物以外，注射剂型可含有药学上可接受的载体例如无菌水、林格氏液和等渗氯化钠溶液，也可根据药物的性质加入适宜的附加剂例如抗氧化剂、缓冲剂和抑菌剂。
另一种示例性的施用途径是局部给药，例如经皮给药(如通过经皮贴剂或离子电渗装置给药)、眼内给药或者鼻内或吸入给药。用于经皮给药的剂型可以为局部凝胶剂、喷雾剂、软膏剂和霜剂。除活性化合物以外，局部剂型可含有能够提高该活性化合物通过皮肤或其它作用区域的吸收或渗透的成分。
另一种示例性的施用途径是直肠给药。用于直肠给药的剂型可以为栓剂。
此外，还可以使用药学领域已知的其它载体材料和给药方式。本发明的融合蛋白、复合物或组合物、或药物组合物可以通过任何公知的制药工艺制备，例如有效的制剂和给药方法。
在另一方面，本发明提供了一种试剂盒，其含有本发明的融合蛋白、复合物、组合物、分离的核酸分子、载体或宿主细胞。在某些实施方案中，所述试剂盒进一步包括用于转染和/或胞内递送(intracellular delivery)的说明书。在某些实施方案中，所述试剂盒用于转染和/或胞内递送货物分子(例如，核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物)。在某些实施方案中，所述细胞是哺乳动物细胞，例如人类细胞。
在另一方面，本发明还涉及本发明的融合蛋白、复合物、组合物、分离的核酸分子、载体或宿主细胞，作为递送试剂(例如转染试剂或胞内递送试剂)的用途。在某些实施 方案中，所述递送试剂用于胞内递送货物分子(例如，核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物)。在某些实施方案中，所述细胞是哺乳动物细胞，例如人类细胞。
在另一方面，本发明提供了一种用于将分子递送入细胞的方法，其包括使所述细胞与本发明的复合物接触，其中所述复合物包括所述分子。
在某些实施方案中，所述细胞与所述复合物的接触在体内实施。
在某些实施方案中，所述细胞与所述复合物的接触离体实施。
在某些实施方案中，所述细胞与所述复合物的接触在体外实施。
在某些实施方案中，所述细胞是真核细胞，例如哺乳动物细胞，例如人类细胞。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“分离的”是指，经人工手段从天然状态下获得。如果自然界中出现某一种“被分离”的物质或成分，那么可能是其所处的天然环境发生了改变或从天然环境下离分出该物质，或二者情况均有发生。比如，对于某一活体动物体内天然存在的某种未被分离的多聚核苷酸或多肽而言，从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为“分离的”。术语“分离的”不排除人工或合成的物质的存在，也不排除不影响物质活性的其它不纯物质的存在。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载 体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如 Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂，稳定剂等。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性(例如对PSD-95泛素化的抑制活性)，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。
发明的有益效果
本发明的递送系统能够显著提高货物分子从内吞小泡的释放，从而显著提升货物分子的细胞质递送效率，使货物分子充分发挥其相应的生物学功能。因此，本发明的递送系统提供了用于影响细胞的生物学机制和途径的有效手段，可用于研究、治疗、诊断等诸多领域，具有广阔的应用前景及临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员 来说将变得显然。
附图说明
图1显示了实施例1中Split-GFP系统检测内吞小泡逃逸效率的原理示意图。
图2显示了实施例1中递送系统-GFPβ1-10蛋白复合物的结构示意图。
图3-4显示了实施例1中递送系统-GFPβ1-10复合物的SDS-PAGE结果。
图5显示了实施例1中递送系统转导GFPβ1-10的流式细胞仪分析结果。
图6显示了实施例1中递送系统转导GFPβ1-10的荧光显微镜观察结果。
图7显示了实施例1中包含酶切位点突变的递送系统转导GFPβ1-10的流式细胞仪分析结果。
图8显示了实施例1中经递送系统转导后的胞内发生切割及未切割/完整的GFPβ1-10相对比例及其在胞内存留时间的Western blot检测结果。
图9显示了实施例1中包含酶切位点突变的递送系统转导后的胞内发生切割及未切割/完整的GFPβ1-10相对比例的Western blot检测结果。
图10显示了实施例1中包含酶切位点但不存在pH敏感肽的递送系统转导后流式细胞仪分析结果。
图11显示了实施例1中包含酶切位点但不存在pH敏感肽的递送系统转导后的胞内发生切割及未切割/完整的GFPβ1-10相对比例的Western blot检测结果。
图12显示了实施例2中递送系统-ZFP9复合物的结构示意图。
图13显示了实施例2中递送系统-ZFP9复合物的SDS-PAGE结果。
图14显示了实施例2中含有ZFP9结合位点的真核表达质粒图谱。
图15-16显示了实施例2中递送系统转导ZFP9的流式细胞仪分析结果。
图17显示了实施例3中递送系统-Ppm1b复合物的结构示意图。
图18显示了实施例3中递送系统-Ppm1b复合物的SDS-PAGE结果。
图19显示了实施例3中递送系统-Ppm1b复合物对TNF-α诱发的细胞坏死比率的影响的流式细胞仪分析结果。
图20显示了实施例4中递送系统-Cas9复合物的结构示意图。
图21显示了实施例4中递送系统-Cas9复合物的SDS-PAGE结果。
图22显示了实施例4中应用HEK293T-RFP reporter细胞检测CRISPR/Cas9编辑效率的原理示意图。
图23显示了实施例4中RFP repoter慢病毒质粒图谱。
图24显示了实施例4中递送系统-Cas9复合物的基因编辑效率的流式细胞仪分析结果。
图25显示了实施例5中基于adapter方式的递送系统原理。
图26显示了实施例5中基于adapter方式的递送系统重组蛋白相关克隆设计。
图27显示了实施例5中基于adapter方式的递送系统重组蛋白纯化结果。
图28显示了实施例5中基于Split-GFP小泡逃逸体系评价基于adapter方式的递送系统的递送效果。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
SEQ ID NO 描述
1 弗林蛋白酶识别序列-1
2 弗林蛋白酶识别序列-2
3 弗林蛋白酶识别序列-3
4 弗林蛋白酶识别序列-4(Ne)
5 编码Ne的核酸序列
6 CTSL识别序列N
7 编码N的核酸序列
8 INF7
9 编码INF7的核酸序列
10 Tat(48-60)
11 编码Tat(48-60)的核酸序列
12 融合蛋白TIN
13 融合蛋白TINe
14 融合蛋白TINNe
15 NLS
16 CTSL识别序列Na
17 CTSL识别序列Nb
18 Furin识别序列Nc
19 Furin识别序列Nd
20 CTSD识别序列Nf
21 突变的N
22 突变的Ne
23 GFPβ1-10-NLS
24 编码GFPβ1-10-NLS的核酸序列
25 编码Histone-H3的核酸序列
26 编码GFPβ11的核酸序列
27 ZFP9-NLS
28 编码ZFP9-NLS的核酸序列
29 编码BFP的序列
30 ZFP9结合位点序列
31 Ppm1b
32 编码Ppm1b的核酸序列
33 Cas9-NLS
34 编码Cas9-NLS的核酸序列
35 编码dsRed的核酸序列
36 编码mCherry的核酸序列
37 sgRNA的识别位点DNA序列
38 流感病毒HA2
39 KALA
40 GALA
41 Melittin
42 Penetratin
43 HIV-TAT(47-57)
44 HIV-1Rev(34-50)
45 VP22
46 Transportan
47 Pep-1
48 Pep-7
49 亮氨酸拉链NZ
50 亮氨酸拉链CZ
51 编码亮氨酸拉链NZ的核酸序列
52 编码亮氨酸拉链CZ的核酸序列
53 TrxA氨基酸序列
54 TrxA核酸序列
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons, Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
以下实施例涉及的主要试剂的来源如下：
克隆构建相关试剂所需材料如下：DNA聚合酶(TaKaRa,R040A)，DNA回收试剂盒(TianGen,DP214-03)，质粒小提试剂盒(TianGen,DP103-03)，质粒大提试剂盒(QIAGEN,12663)，Gibson装配预混液5管(NEB,E2611L)，DNA marker(ThmeroFisher，SM0331)，琼脂糖(Biowest，BW-R0100)，
蛋白大量表达所需材料如下：蛋白胨(BiSIGMA-ALDRICH，T7293-1KG)，酵母粉(OXOID，LP0021B)，氯化钠(西陇化工，10011012AR)，IPTG(Inalco，1758-1400)
蛋白纯化所需介质如下：SP SEPHAROSE FAST FLOW(GE Healthcare，17-0729-01)，NI SEPHAROSE(GE Healthcare，17-5268-02)
蛋白纯化及保存所需试剂如下：甘油/丙三醇/C 3H 8O 3(SIGMA-ALDRICH,G5516),KCl(西陇化工，1002007),Na 2HPO 4·12H 2O(西陇化工，1001067AR)，KH 2PO 4(西陇化工，1002048AR500)，咪唑(SIGMA-ALDRICH,V900153)，Tris base(Seebio，183995)，葡萄糖(西陇化工，1064008AR500)，BCA蛋白浓度测定试剂盒(Thermo Scientific，23227)；
细胞培养所需试剂：FBS(GIBCO，10099-133)，DMEM(GIBCO，11965092)，胰蛋白酶(AMRESCO，0458)；
慢病毒包装及感染所需试剂：慢病毒包装质粒：pCMV-VSV-G(Addgene，8454)，pRSV-Rev(Addgene，12253)，pMDLg/pRRE(Addgene，12251)；X-tremeGENE转染试剂(Roche，06366244001)，Puromycin(InvivoGen，ant-pr-5)，Blasticidin(InvivoGen，ant-bl-5b)，polybrene(Santa Cruz，sc-134220)；
实验中所用的GFPβ1-10,ZFP9,Ppm1b,dsRed,mCherry，Histone-H3相关质粒均由公司合成(生工生物)，用于扩增Cas9序列的质粒pCasKP-hph(Addgene，117232)；
其他试剂：TNF-α(Novoprotein，CF09),PI(ThmeroFisher，P3566)
细胞系：HEK-293T(人肾上皮细胞)，L929(小鼠成纤维细胞)均购自ATCC。
实施例1：基于Split-GFP系统评价递送系统的内吞小泡逃逸效率
在Split-GFP系统中，GFP的11个β折叠被分拆成大片段(β1-10)和小片段(β11)，二者均失去了其荧光活性，但如果相遇则可以自发缔合并恢复GFP的荧光性能。基于此，我们构建了稳定表达Histone-β11的HEK293T细胞，同时将带有入核信号(NLS)的GFPβ1-10作为Cargo与拟评价的入胞递送系统融合表达，然后转导该稳定细胞系。当递送系统转导GFPβ1-10时，只有成功从内吞小泡逃逸并进入到细胞质或者细胞核后才能与GFPβ11结合生成完整的GFP，因此通过GFP的比例及相对荧光强度可对内吞小泡逃逸效率进行评估(图1)。
1.1递送系统-GFPβ1-10蛋白复合物表达载体的构建
构建含有TAT(SEQ ID NO:10)、INF7(SEQ ID NO:8)、蛋白酶切割位点(表2)和携带核定位信号(NLS)的货物分子GFPβ1-10(SEQ ID NO:23)的重组蛋白表达载体，各重组蛋白的结构示意图如图2所示，从C端到N端各组分氨基酸序列如下表3所示。所述构建方法如下：首先，通过PCR扩增得到编码递送系统中的TAT、INF7、N、Na、Nb、Nc、Nd、Ne、Nf、突变的N、突变的Ne、货物分子GFPβ1-10的核酸序列，并通过多轮PCR将各部分连接，并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET21b(+)对应的NdeI酶切位点上游的重叠序列，通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI，BamHI双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-21b(+)上。
表2：蛋白酶识别序列
Figure PCTCN2020101424-appb-000001
表3：递送系统-货物分子复合物所包含的组分
Figure PCTCN2020101424-appb-000002
1.2递送系统-GFPβ1-10复合物的表达及纯化：
将1.1中所述的表达质粒转化入表达菌株E.coli BL21(DE3)；从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜，然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中，37℃，180rpm培养到菌液OD 600在0.6左右，接着加入诱导剂IPTG至终浓度为0.2mM，25℃诱导8h；诱导表达结束后，4℃，7000g离心10min后收集菌体；接着用10ml蛋白纯化平衡缓冲液(50ml甘油，8g NaCl，0.201g KCl，1.44g Na 2HPO 4，0.24g KH 2PO 4溶于1L双蒸水中)重悬菌体，进行超声破碎。接着离心取上清液，并上样到蛋白纯化系统的聚组氨酸蛋白纯化柱上；然后利用蛋白纯化系统用蛋白纯化洗脱缓冲液(50ml甘油，8g NaCl，0.201g KCl，1.44g Na 2HPO 4，0.24g KH 2PO 4，17g咪唑溶于1L双蒸水中)洗脱目的蛋白。蛋白浓度可以 根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。各蛋白的SDS-PAGE结果如图3-4所示。
1.3 HEK293T-GFPβ11细胞系的构建
1.3.1 GFPβ11细胞系慢病毒质粒的构建:
Histone-H3的编码序列(SEQ ID NO:25)通过PCR扩增得到，GFPβ11编码序列(SEQ ID NO:26)较短直接设计在上游引物中，通过多轮PCR将各组分连接，并在最后一轮PCR过程中通过上游引在片段的5’端引入Hind III酶切位点及其与Lenti载体对应的Hind III酶切位点上游的重叠序列，通过下游引在片段的3’端引入酶切位点BamHI及其与Lenti载体对应的BamHI酶切位点下游的重叠序列。Lenti质粒通过Hind III，BamH I双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切后的Lenti载体上。
1.3.2慢病毒包装、感染及抗性筛选细胞系：
将HEK-293T细胞接种到6孔板中，过夜培养，在质粒转染之前保证每孔的细胞数约为2*10 7/ml左右；转染之前，细胞更换为无血清DMEM培养基；在300μl无血清DMEM分别加入1.5μg Lenti重组质粒，0.75μg pMDL质粒，0.45μg pVSV-G质粒,0.3μg pREV(质量比为5:3:2:1),缓慢吹匀后，加入9μl(1:3)的X-tremeGENE转染试剂，缓慢吹匀，室温静置15min，滴加入细胞上清中，8h后更为换为含10％FBS的DMEM继续培养；60h后收取培养上清待感染。
将HEK-293T细胞接种到12孔板中，过夜培养，在慢病毒感染之前保证每孔的细胞数约为2*10 6/ml(50％密度)左右；弃去原有细胞培养上清，加入300μl慢病毒(moi＝3)及700μl 10％FBS DMEM，按10μg/ml的浓度加入polybrene，细胞板无菌条件下2500rpm离心30min后继续培养。
慢病毒感染48h后按1/3的比例进行细胞传代，同时按2.5μg/ml的浓度加入puromycin进行抗性筛选；将筛选得到的阳性细胞进行克隆化，得到HEK-293T-Hitone-GFPβ11的单克隆细胞株。
1.4 Split-GFP系统检测递送系统-GFPβ1-10复合物的内吞小泡逃逸效率
将1.3中获得的HEK-293T-Hitone-GFPβ11细胞系接种到12孔板中，过夜培养，蛋白处理前保证每孔的细胞数约为5*10 6/ml左右；用无血清DMEM培养基润洗三次细胞， 在无血清培养基的环境下加入100μl/5μM 1.2中获得的递送系统-GFPβ1-10复合物，孵育3h，用肝素溶液洗涤三次去除细胞表面吸附且尚未内吞入胞的蛋白后，更换为含10％FBS DMEM培养基继续培养，在12h进行荧光显微镜观察及流式分析绿色荧光蛋白表达情况。
流式细胞仪分析结果如图5所示，结果显示，在TAT基础上加入pH敏感肽INF7后细胞平均荧光强度有所提升，证明了pH敏感肽对内吞小泡囊膜的破膜作用；特别地，在TAT-INF7的基础上加入CTSL或Furin的酶切位点后，细胞荧光强度进一步上升，其中CTSL的酶切位点N以及Furin的酶切位点Ne效果最为显著。荧光显微镜观察如图6所示，结果显示，将上述两种酶切位点联合(TINNe-GFPβ1-10)能够进一步显著提升内吞小泡逃逸效率。
在N或Ne酶切位点中的关键位点引入突变以获得包含突变的递送系统-货物分子复合物，其中，突变的N的序列如SEQ ID NO:21所示，突变的Ne的序列如SEQ ID NO:22所示；并比较突变的递送系统-货物分子复合物(Mut)与不含突变的递送系统-货物分子复合物(WT)的转染效率。结果如图7所示，当将酶切位点中的关键氨基酸突变后，无论是单个酶切位点，还是双酶切位点联用，均失去了其增强小泡逃逸效率的作用。以上结果表明，CTSL及Furin酶切位点确实可以显著提升Cargo在小泡中的逃逸效率。
用肝素溶液洗涤三次去除细胞表面吸附且尚未内吞入胞的蛋白后，收集转导开始后不同时间节点的细胞，裂解细胞提取蛋白并进行SDS-PAGE电泳，随后利用识别GFPβ1-10的单抗(Abcam，ab32146)进行western blot检测，以对胞内蛋白的酶切情况与留存情况进行分析。结果如图8所示，在6h前的各个时间节点，检测到的胞内蛋白总量在各组细胞间相当，表明pH敏感肽和酶切位点并不增加Cargo的细胞内吞效率；含酶切位点的蛋白(TIN-、TINe-、TINNe-)在30min时即已出现酶切现象，3h后酶切后的蛋白量不再增加，此时，单酶切位点蛋白约40％出现酶切，而双酶切位点蛋白约70％出现酶切；此后，没有酶切位点的蛋白(T-、TI-)以及酶切位点未被酶切的完整蛋白由于被导向溶酶体开始快速降解，至12h时相应条带几乎消失不见，而酶切后与TAT-INF7分离的蛋白仍有留存，且TINNe组存留最多。以上结果与流式细胞术检测结果一致，酶切效率越高，细胞内未被降解的留存GFPβ1-10-NLS越多，其进入核内与GFPβ11缔合成的完整GFP越多，绿色荧光强度越高。
进一步，对上述在N或Ne酶切位点中包含突变的递送系统-货物分子复合物转导细胞3h后的酶切情况分析进行western blot检测。结果如图9所示，突变后的递送系统-货 物分子复合物(TINm-、TINem-和TINNem-)无法实现在胞内的切割。因此结合图7所示的流式结果可知，无法实现酶切及之后的逃逸是突变后的递送系统-货物分子复合物失去高效的递送效果的原因，也进一步确认了CTSL,Furin酶切位点的在递送系统中的关键作用。
此外，还对缺失pH敏感肽组分的递送系统-复合物(TNNe-GFPβ1-10-NLS)的递送效率进行了流式检测，结果如图10所示。我们发现其转导12h后的平均荧光强度和T-GFPβ1-10-NLS基本一致，说明即使存在酶切位点，但缺少pH敏感肽也无法实现高效递送。同时通过western blot检测发现，有无pH敏感肽对递送系统-复合物在胞内的酶切效率并无影响(图11)。结合以上结果，我们证实了只有pH敏感肽与特定酶切位点在递送系统的同时存在才可实现最终的高效递送。
实施例2：递送系统在转导锌指蛋白ZFP中的应用
2.1递送系统-锌指蛋白ZFP9复合物表达载体的构建
构建含有TAT、INF7、蛋白酶切割位点、携带核定位信号(NLS)的货物分子ZFP9(SEQ ID NO:27)的重组蛋白表达载体，各重组蛋白的结构示意图如图12所示，各组分氨基酸序列如下表4所示。所述构建方法如下：首先，通过PCR扩增得到编码递送系统中的TAT、INF7、蛋白酶切割位点、货物分子ZFP9的核酸序列，并通过多轮PCR将各部分连接，并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET-21b(+)对应的NdeI酶切位点上游的重叠序列，通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI，BamHI双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-21b(+)上。
表4：递送系统-货物分子复合物所包含的组分
Figure PCTCN2020101424-appb-000003
2.2递送系统-ZFP9复合物的表达、纯化
将2.1中所述的表达质粒转化入表达菌株E.coli BL21(DE3)；从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜，然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中，37℃，180rpm培养到菌液OD 600在0.6左右，接着添加诱导IPTG至终浓度为0.2mM，25℃诱导8小时；诱导表达结束后，4℃，7000g离心10min后收集菌体，取部分菌体检测蛋白质诱导表达情况；接着用10ml蛋白纯化平衡缓冲液(50ml甘油，3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中，调pH至8.0)重悬菌体，进行超声破碎。接着离心取上清液，并上样到AKTA蛋白纯化系统的Sulphopropyl(SP)阳离子交换柱上；然后利用平衡缓冲液与高盐洗脱液(50ml甘油，116.88g NaCl，3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中，调pH至8.0)之间按不同比例来进行梯度洗脱得到目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。各蛋白的SDS-PAGE结果如图13所示。
2.3含有ZFP9结合位点的真核表达质粒的构建
构建含有蓝色荧光蛋白(BFP)编码序列和ZFP9结合位点的表达载体，其结构示意图如图14所示。蓝色荧光蛋白编码序列(SEQ ID NO:29)及ZFP9结合位点6*binding sites(6个结合位点)(SEQ ID NO:30)序列通过PCR扩增得到，通过两轮PCR将二者连接，并在第二轮PCR过程中通过上游引在片段的5’端引入Hind III酶切位点及其与pTT5载体对应的Hind III酶切位点上游的重叠序列，通过下游引在片段的3’端引入酶切位点BamHI及其与pTT5载体对应的BamHI酶切位点下游的重叠序列。pTT5质粒通过Hind III，BamH I双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切后的pTT5载体上，从而获得pTT5-BFP-6BS质粒。
2.4递送系统转导锌指蛋白ZFP9的递送效率检测
将HEK293T细胞接种到12孔板中，过夜培养，蛋白处理前保证每孔的细胞数约为5*10 6/ml左右；将100μL/5μM 2.2中获得的递送系统-ZFP9复合物(ZFP9、T-ZFP9、TI-ZFP9、TINNe-ZFP9)和2.3中获得的pTT5-BFP-6BS质粒5μg在37℃共孵育30min充分形成复合物，并以X-tremeGENE转染试剂(Roche)作为阳性对照(5μg质粒与15μl转染试剂混合后在无血清条件下转染细胞，并在8h后更换为10％FBS DMEM继续培养)；用无血清DMEM培养基润洗三次细胞，后加入复合物孵育3h，用 肝素溶液洗涤三次去除细胞表面吸附且尚未内吞入胞的蛋白后，更换为含10％FBS DMEM培养基继续培养更换有10％FBS DMEM继续培养，分别在换液12h、24h、36h、48h进行流式分析蓝色荧光蛋白表达情况。
流式检测结果如图15-16所示。与ZFP9结合的质粒只有进入细胞核内才能完成转录过程，因此，只有更多的ZFP9的从内吞小泡中逃逸，才能携带结合的质粒进入细胞核内，开始转录过程，进而表达蓝色荧光蛋白。相对于T-ZFP9，pH敏感肽INF7的加入(TI-ZFP9)可以使蓝色荧光比率上升10％左右(p＝0.035)，但仍然处于一个较低的水平，即大部分复合物未能逃逸并入核，而在此基础上，在系统中引入蛋白酶切割位点后(TINNE-ZFP9)，结果显示蓝色荧光细胞比率大幅度上升，在48h时可达到65％左右(p＝0.018)，且能够与Roche转染试剂X-tremeGENE持平。由此可见，在递送系统携带ZFP9/pTT5-BFP-6BS复合物入胞的内吞过程中，CTSL和Furin特异性切割位点N及Ne加入显著促进了所携带的ZFP9的逃逸过程，更多的复合物可以进入核内完成转录，进而表达BFP荧光。
实施例3：递送系统在转导蛋白磷酸酶Ppm1b中的应用
TNF-α与细胞表面受体结合后诱导RIP3磷酸化并形成多蛋白复合物的坏死小体(Necrosome)，坏死小体中的磷酸化RIP3招募并磷酸化Mlkl后细胞进入坏死程序。在此过程中，胞内的蛋白磷酸酶1B(Ppm1b)可通过使RIP3去磷酸化从而抑制细胞程序性坏死(Necroptosis)。鉴于目前已发现细胞程序性坏死与炎性疾病、缺血再灌注损伤、神经退行性疾病等多种疾病的发生密切相关，Ppm1b蛋白在治疗上述与细胞程序性坏死相关的疾病方面展现出巨大潜力。
3.1递送系统-Ppm1b蛋白复合物表达载体构建
构建含有TAT、INF7、蛋白酶切割位点、货物分子Ppm1b(SEQ ID NO:31)的重组蛋白表达载体，各重组蛋白的结构示意图如图17所示，各组分氨基酸序列如下表6所示。所述构建方法如下：通过PCR扩增得到TAT、INF7、蛋白酶切割位点、Ppm1b的核酸序列，并通过多轮PCR将各部分连接，并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET-21b(+)对应的NdeI酶切位点上游的重叠序列，通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI，BamHI双酶切处理。通过GIBSON组装方式将带有重叠序列的插入片段连接到酶切载体pET-21b(+)。
表5：递送系统-货物分子复合物所包含的组分
Figure PCTCN2020101424-appb-000004
3.2递送系统-Ppm1b复合物的表达、纯化
将3.1中所述的表达质粒转化入表达菌株E.coli BL21(DE3)；从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜，然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中，37℃，180rpm培养到菌液OD 600在0.6左右，接着添加诱导IPTG至终浓度为0.2mM，25℃诱导8h；诱导表达结束后，4℃，7000g离心10分钟后收集菌体，取部分菌体检测蛋白质诱导表达情况；接着用10ml蛋白纯化平衡缓冲液(50ml C 3H 8O 3，3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中，调pH至8.0)重悬菌体，进行超声破碎。接着离心取上清液，并上样到AKTA蛋白纯化系统的Sulphopropyl(SP)阳离子交换柱上；然后利用平衡缓冲液与高盐洗脱液(50ml C 3H 8O 3，116.88g NaCl，3.6342g Tris(Hydroxymethyl)aminomethane溶于1L双蒸水中，调pH至8.0)之间按不同比例来进行梯度洗脱得到目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。各蛋白的SDS-PAGE结果如图18所示。
3.3递送系统-Ppm1b复合物对TNF-α诱发的细胞发生坏死比率的影响
将L929细胞接种到12孔的细胞培养板中，过夜培养，蛋白处理前保证每孔的细胞数约为2*10 6/ml左右；用无血清DMEM培养基润洗三次细胞，然后在无血清培养基中分别加入100μl/5μM的3.2中获得的递送系统蛋白(Ppm1b、T-Ppm1b、TI-Ppm1b、TINNe-Ppm1b)，孵育3h后，加入用10％FBS DMEM稀释好含20ng/ml TNF-α以及20mM z-VAD 1ml，孵育10h后，收集细胞进行PI染色，流式分析细胞观察细胞坏死的比率。利用慢病毒转导Ppm1b(Lenti-Ppm1b)以及慢病毒(Lenti-vec)作为对照，在HEK-293T细胞上包装收集的带有Ppm1b表达序列的慢病毒和对照慢病毒，感染L929细胞24h后，以使Ppm1b在胞内充分表达，将感染后的L929细胞重新铺版待TNF-α刺 激。
结果如图19所示，相对于单纯的T-Ppm1b，pH敏感肽INF7的加入(TI-Ppm1b)仅能略微抑制细胞坏死，但仍然处于一个较低的水平，而在此基础上，在系统中引入蛋白酶切割位点后(TINNe-Ppm1b)，TINNe组抑制细胞坏死的能力可达到慢病毒组的水平，细胞坏死率可大大降低至约25％。因此，在递送系统携带Ppm1b复合物入胞的内吞过程中，CTSL和Furin特异性位点N及Ne的加入显著促进了所携带的Ppm1b的逃逸过程，更多的Ppm1b进入细胞质内，抑制RIP3的磷酸化过程，降低细胞坏死比率。
实施例4：递送系统在基因编辑工程酶Cas9中的应用
CRISPR/Cas9基因编辑系统是通过sgRNA结合Cas9蛋白，通过sgRNA特异性识别靶向位点，Cas9结合并切割DNA双链分子，通过非同源性末端重组或者同源性末端修复实现对靶向基因的编辑。该系统中Cas9必须进入细胞核内完成其功能，基于此我们将递送系统融合表达Cas9蛋白，实现在真核细胞上的基因编辑。
4.1递送系统-Cas9蛋白复合物表达载体的构建
构建含有TAT、INF7、蛋白酶切割位点、携带核定位信号(NLS)的货物分子Cas9(SEQ ID NO:33)的重组蛋白表达载体，各重组蛋白的结构示意图如图20所示，各组分氨基酸序列如下表6所示。所述构建方法如下：通过PCR扩增得到编码TAT、INF7、蛋白酶切割位点N及Ne、Cas9的核酸序列，并通过多轮PCR将各部分连接，并在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET-21b(+)对应的NdeI酶切位点上游的重叠序列，通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列(overlap)。pET-21b(+)质粒通过NdeI，BamHI双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-21b(+)上。
表6：递送系统-货物分子复合物所包含的组分
Figure PCTCN2020101424-appb-000005
4.2递送系统-Cas9复合物的表达及纯化
镍柱初纯：将4.1中所述的表达质粒转化入表达菌株E.coli BL21(DE3)；从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜，然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中，37℃，180rpm培养到菌液OD 600在0.6左右，接着加入诱导剂IPTG至终浓度为0.2mM，25℃诱导8h；诱导表达结束后，4℃，7000g离心10分钟后收集菌体；接着用10ml蛋白纯化平衡缓冲液(5％甘油，30mM TB8.0，50mM丙三醇，500mM氯化钠，25mM葡萄糖)重悬菌体，进行超声破碎。接着离心取上清液，并上样到蛋白纯化系统的聚组氨酸蛋白纯化柱上；然后利用蛋白纯化系统用蛋白纯化洗脱缓冲液(5％甘油30mM TB8.0，50mM丙三醇，500mM氯化钠，25mM葡萄糖，250mM咪唑)洗脱目的蛋白。
阳离子交换柱精纯：将镍柱初纯收集的目的蛋白透析至平衡缓冲液(30mM TB8.0，50mM丙三醇，25 0mM氯化钠，25mM葡萄糖，调节pH至7.2)中，并上样到AKTA蛋白纯化系统的Sulphopropyl(SP)阳离子交换柱上；然后利用平衡缓冲液与高盐洗脱液(30mM TB8.0，50mM丙三醇，2M氯化钠，25mM葡萄糖，调节pH至7.2)之间按不同比例来进行梯度洗脱得到目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。各蛋白的SDS-PAGE结果如图21所示。
4.3 HEK293T-RFP reporter细胞系的构建
Cas9和sgRNA结合后可以特异性识别靶向位点，在不提供donor的情况下，会发生非同源性末端重组。因此通过慢病毒感染的方式将sgRNA识别位点及两个不在阅读框内的红色荧光蛋白基因(dsRed，mCherry之间差一个G)整合到HEK-293T细胞的基因组上，如果Cas9导致sgRNA识别位点上的DNA序列方生同源性末端重组，则会导致本来不在阅读框内的红色荧光蛋白基因进入阅读框而表达，而使细胞从无荧光变为红色荧光，通过是否产生红色荧光及红色荧光细胞的数目可以对递送系统转导基因工程酶Cas9的效率进行评价(图22)。
4.3.1 RFP repoter细胞系慢病毒质粒的构建
通过PCR扩增得到dsRed(SEQ ID NO:35)及mCherry的编码序列(SEQ ID NO:36)，sgRNA的识别位点DNA序列(SEQ ID NO:37)较短直接设计在引物中，通过多轮PCR将各组分连接，并在最后一轮PCR过程中通过上游引在片段的5’端引入Hind III酶切位点及其与Lenti载体对应的Hind III酶切位点上游的重叠序列，通过下游引在 片段的3’端引入酶切位点BamHI及其与Lenti载体对应的BamHI酶切位点下游的重叠序列。Lenti质粒通过Hind III，BamH I双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切后的Lenti载体上，构建成功的质粒图谱(图23)。
4.3.2慢病毒包装、感染及细胞系抗性筛选
将HEK293T细胞接种到6孔板中，过夜培养，在质粒转染之前前保证每孔的细胞数约为2*10 7/ml左右；转染之前，细胞更换为无血清DMEM培养基；在300μl无血清DMEM分别加入1.5μg Lenti重组质粒(RFP reporter)，0.75μg pMDL质粒，0.45μg pVSV-G质粒，0.3μg pREV质粒(质量比为5:3:2:1)，缓慢吹匀，静置5min后，加入9μl的X-tremeGENE转染试剂，缓慢吹匀，室温静置15min后，加入至细胞上清内，8h后更换为含有10％FBS DMEM继续培养；60h后收取培养上清，4℃保存。
将HEK293T细胞接种到12孔板中，过夜培养，在慢病毒感染之前保证每孔的细胞数约为2*10 6/ml(50％密度)左右；弃去细胞培养上清，加入300μl慢病毒(moi＝3)及700μl 10％FBS DMEM，按10μg/ml的浓度加入polybrene，无条件下2500rpm离心30min后继续培养。
慢病毒感染48h后按1/3的比例进行细胞传代，同时按2.5μg/ml的浓度加入puromycin进行抗性筛选；将筛选得到的阳性细胞进行克隆化，得到HEK293T-RFP reporter的单克隆细胞株。
4.3.3 GM3-gRNA转录质粒的构建
gRNA的导入采用转录质粒转染后在细胞内转录成为gRNA的方式，gRNA所对应的DNA序列通过引物退火互搭产生，在引物设计的过程中直接引入AflII酶切位点的黏性末端；gRNA-cloning载体利用AflII单酶切处理；载体和插入片段利用各自的黏性末端通过T4DNA连接酶进行连接。
4.4递送系统-Cas9复合物的编辑效率评价
gRNA转录质粒转染：将HEK-293T-RFP reporter细胞系接种到12孔板中，过夜培养，转染前保证每孔的细胞数约为2.5*10 6/ml左右；转染前细胞更换为无血清DMEM；在100μl无血清DMEM中加入1μg gRNA-GM3转录质粒，缓慢吹匀，静置5min，加入3μl X-tremeGENE转染试剂，缓慢吹匀后静置15min，加入至细胞上清中，8h后更换为10％FBS DMEM。
递送系统转导Cas9：在gRNA转录质粒转染12h后(换为有血清DMEM后4h)，用无血清DMEM润洗三次细胞，在无血清DMEM的环境下加入5μM的4.2中获得的 递送系统-Cas9复合物孵育3h；更换为10％FBS DMEM继续培养，在48h进行观察及流式分析红色荧光蛋白表达情况。
流式结果如图24所示，T-Cas9转导后，红色荧光细胞比例较低(3.7％)，即大部分Cas9蛋白未能逃逸并入核；pH敏感肽INF7的加入(TI-Cas9)能够增加红色荧光细胞比例至9％；而在此基础上，在系统中引入蛋白酶切割位点后(TINNE-Cas9)，表达红色荧光蛋白的细胞数目显著上升，在48h可达到14％左右。因此，在递送系统携带Cas9入胞的内吞过程中，CTSL和Furin特异性位点N及Ne的加入显著促进了所携带的Cas9的逃逸过程，更多的Cas9在入核信号的作用下进入细胞核，在与胞内转录形成的GM3sgRNA结合后，特异性识别靶标序列，并在该位点附近发生同源性末端修复，从而使红色荧光基因进入阅读框并表达。
实施例5：基于非共价连接的递送系统的建立
本发明的融合蛋白与货物的连接方式除融合表达之外，还可以通过蛋白结构域进行非共价连接，我们称之为Adapter作用对，如具有较强相互作用力的异源二聚体亮氨酸拉链(图25)。两个反向平行的亮氨酸拉链结构域因其互相嵌合的空间特点以及电荷排布，可以自发地结合形成寡聚体。据研究报道，NZ与CZ就是这样一对可以相互结合的亮氨酸拉链。我们尝试将NZ结构域与本发明的入胞递送系统融合表达(TINNe-NZ)，CZ结构域与带有入核信号(NLS)的GFPβ1-10融合表达作为Cargo(CZ-GFPβ1-10-NLS)。将上述两种蛋白混合孵育相互结合后，转导稳定表达Histone-β11的HEK293T细胞，通过GFP的比例及相对荧光强度可对内吞小泡逃逸效率进行评估。从而评估NZ-CZ这对adapter连接融合蛋白与货物的可行性。
5.1递送系统-NZ融合蛋白及CZ-GFPβ1-10融合蛋白表达载体的构建
基于pET32a载体，构建含有TAT、INF7、蛋白酶切割位点和NZ结构域的重组蛋白的表达载体，以及构建携带CZ结构域、核定位信号(NLS)的货物分子GFPβ1-10的重组蛋白表达载体，其中添加TrxA作为促溶标签。重组蛋白的结构示意图如图26所示，递送系统氨基酸序列如下表所示。所述构建方法如下：首先，以如前所述载体为模板，通过PCR扩增得到编码递送系统中的TAT、INF7、N、Ne及NZ序列，以及CZ序列、货物分子sGFPβ1-10的核酸序列，对于TrxA-TINNe-NZ，在最后一轮PCR过程中通过上游引物在片段的5’端引入BamHI酶切位点及其与pET-32a(+)对应的BamHI酶切位点上游的重叠序列，通过下游引在片段的3’端引入HIindIII酶切位点及其与pET-32a(+)对 应的HIindIII酶切位点下游的重叠序列。pET-32a(+)质粒通过BamHI,HIindIII双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-32a(+)上；对于CZ-GFPβ1-10，在最后一轮PCR过程中通过上游引在片段的5’端引入NdeI酶切位点及其与pET21b(+)对应的NdeI酶切位点上游的重叠序列，通过下游引在片段的3’端引入BamHI酶切位点及其与pET-21b(+)对应的BamHI酶切位点下游的重叠序列。pET-21b(+)质粒通过NdeI，BamHI双酶切处理。通过GIBSON组装将带有重叠序列的插入片段连接到酶切载体pET-21b(+)上。
表7：递送系统所包含的组分
Figure PCTCN2020101424-appb-000006
5.2递送系统-NZ融合蛋白及CZ-GFPβ1-10融合蛋白的表达及纯化
将5.1中所述的表达质粒转化入表达菌株E.coli BL21(DE3)；从转化的平板上挑选单菌落接种到含氨苄抗性的5ml LB液体培养基中培养过夜，然后取1ml过夜培养的菌液转接到含氨苄抗性的500ml LB液体培养基中，37℃，180rpm培养到菌液OD 600在0.6左右，接着加入诱导剂IPTG至终浓度为0.2mM，25℃诱导8h；诱导表达结束后，4℃，7000g离心10min后收集菌体；接着用10ml蛋白纯化平衡缓冲液(50ml甘油，8g NaCl，0.201g KCl，1.44g Na 2HPO 4，0.24g KH 2PO 4溶于1L双蒸水中)重悬菌体，进行超声破碎。接着离心取上清液，并上样到蛋白纯化系统的聚组氨酸蛋白纯化柱上；然后利用蛋白纯化系统用蛋白纯化洗脱缓冲液Elution buffer 2#(50ml甘油，8g NaCl，0.201g KCl，1.44g Na 2HPO 4，0.24g KH 2PO 4，17g咪唑溶于1L双蒸水中)洗脱目的蛋白。蛋白浓度可以根据分光光度计或BCA蛋白浓度测定试剂盒进行测定。每个纯化后的融合蛋白分装后-20℃保存。各蛋白的SDS-PAGE结果如图27所示。
5.3 Split-GFP系统检测基于adapter的递送系统的递送效率
将上文中获得的HEK-293T-Hitone-GFPβ11细胞系接种到12孔板中，过夜培养，蛋白处理前保证每孔的细胞数约为5*10 6/ml左右；用无血清DMEM培养基润洗三次细胞，对照组及实验组设置及蛋白(5.2中所获得)用量如下表所示，其中，在与细胞孵育前，对 照组2及实验组中的两种蛋白在无血清培养基中室温混合10min。在无血清培养基的环境下孵育3h，用肝素溶液洗涤三次去除细胞表面吸附且尚未内吞入胞的蛋白后，更换为含10％FBS DMEM培养基继续培养，在12h进行荧光显微镜观察及流式分析绿色荧光阳性细胞的比例及MFI。
流式细胞仪分析结果如图28所示，结果显示，两个对照组(CZ-GFPβ1～10/TrxA-TINNe+CZ-GFPβ1～10)的MFI均低于5，其中对照组2(TrxA-TINNe+CZ-GFPβ1～10)的MFI略高于对照组1，我们推测是因为TrxA-TINNe本身与CZ-GFPβ1～10存在非特异性的吸附导致的，从而通过TINNe的作用将货物CZ-GFPβ1～10带入细胞内并发生内吞小泡逃逸。而TrxA-TINNe-NZ+CZ-GFPβ1～10组的MFI值接近20，显著高于对照组，说明了在NZ和CZ的作用下，TrxA-TINNe与GFPβ1～10发生一定程度的结合，并通过TINNe的作用，使GFPβ1～10进入细胞并发生逃逸。尽管adpater连接方式的荧光强度仍低于融合表达连接方式的TINNe-GFPβ1～10，但该实验证实了基于NZ-CZ或者类似的adapter，可以作为递送系统与货物的连接方式，并能够有效递送货物进入细胞及从小泡中逃逸。
表8：对照组及实验组设置
Figure PCTCN2020101424-appb-000007
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (27)

  1. 融合蛋白，其包含细胞穿膜肽、pH敏感融合肽以及蛋白酶识别序列，其中，所述蛋白酶选自弗林蛋白酶和/或溶酶体半胱氨酸蛋白酶。
  2. 权利要求1所述的融合蛋白，其中，所述弗林蛋白酶识别序列包含R-X 1-X 2-R(SEQ ID NO:1)，其中，X 1为任意氨基酸，X 2为K或R；
    优选地，所述弗林蛋白酶识别序列包含R-R-X 1-X 2-R(SEQ ID NO:2)；
    优选地，所述弗林蛋白酶识别序列包含SEQ ID NO:3所示的序列；
    优选地，所述弗林蛋白酶识别序列包含SEQ ID NO:4所示的序列。
  3. 权利要求1或2所述的融合蛋白，其中，所述溶酶体半胱氨酸蛋白酶选自组织蛋白酶B、组织蛋白酶C、组织蛋白酶X、组织蛋白酶S、组织蛋白酶L、组织蛋白酶D或组织蛋白酶H；
    优选地，所述溶酶体半胱氨酸蛋白酶是组织蛋白酶L；
    优选地，所述组织蛋白酶L识别序列包含SEQ ID NO:6所示的序列。
  4. 权利要求1-3任一项所述的融合蛋白，其中，所述蛋白酶识别序列包含弗林蛋白酶识别序列和组织蛋白酶L识别序列；
    优选地，所述蛋白酶识别序列包含SEQ ID NO:3和SEQ ID NO:6；
    优选地，所述蛋白酶识别序列包含SEQ ID NO:4和SEQ ID NO:6。
  5. 权利要求1-4任一项所述的融合蛋白，其中，所述pH敏感融合肽选自流感病毒HA2或其突变体(例如INF7，KALA或GALA)、蜂毒素(Melittin)，及其任意组合；
    优选地，所述pH敏感融合肽包含INF7；
    优选地，所述pH敏感融合肽包含如SEQ ID NO:8所示的序列。
  6. 权利要求1-5任一项所述的融合蛋白，其中，所述细胞穿膜肽选自穿透素(Penetratin)、Tat衍生肽(例如，Tat(48-60)或Tat(47-57))、Rev(34-50)、VP22、转运肽(transportan)、Pep-1、Pep-7，及其任意组合；
    优选地，所述细胞穿膜肽包含Tat衍生肽，例如Tat(48-60)；
    优选地，所述细胞穿膜肽包含如SEQ ID NO:10所示的序列。
  7. 权利要求1-6任一项所述的融合蛋白，其中，所述融合蛋白从N端至C端包含所述pH敏感融合肽、细胞穿膜肽和蛋白酶识别序列；或者，所述融合蛋白从N端至C端包含所述细胞穿膜肽、pH敏感融合肽和蛋白酶识别序列；
    优选地，所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列，或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列。
  8. 权利要求1-7任一项所述的融合蛋白，其中，所述融合蛋白包含SEQ ID NOs:12-14任一项所示的序列。
  9. 权利要求1-8任一项所述的融合蛋白，其中，所述融合蛋白进一步包含特异性结合序列，所述特异性结合序列允许另外的分子(例如多肽、蛋白或核酸)与其发生特异性结合；
    优选地，所述特异性结合序列包含亮氨酸拉链，并且所述亮氨酸拉链能够与其反向序列形成异源二聚体；
    优选地，所述特异性结合序列包含亮氨酸拉链NZ或CZ；
    优选地，所述特异性结合序列包含SEQ ID NO:49或50所示的序列；
    优选地，所述特异性结合序列位于所述蛋白酶识别序列的C端。
  10. 复合物，其包含权利要求1-9任一项所述的融合蛋白，以及货物分子；
    优选地，所述货物分子选自核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物；
    优选地，所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合；
    优选地，所述货物分子包含可检测的标记；
    优选地，所述货物分子包含表位标签、报告基因序列和/或核定位信号(NLS)序列。
  11. 权利要求10所述的复合物，其中，所述融合蛋白与货物分子是融合的，所述货物分子是肽或蛋白质；
    优选地，所述货物分子融合至所述融合蛋白的C端；
    优选地，所述复合物包含单链多肽，所述单链多肽从N端至C端包含所述pH敏感融合肽、细胞穿膜肽、蛋白酶识别序列和货物分子；优选地，所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列，或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列；
    优选地，所述复合物包含单链多肽，所述单链多肽从N端至C端包含所述细胞穿膜肽、pH敏感融合肽、蛋白酶识别序列和货物分子；优选地，所述蛋白酶识别序列从N端至C端包含所述弗林蛋白酶识别序列和组织蛋白酶L识别序列，或者从N端至C端包含所述组织蛋白酶L识别序列和弗林蛋白酶识别序列。
  12. 权利要求10所述的复合物，其中，所述融合蛋白与货物分子化学偶联；
    优选地，所述化学偶联通过二硫键、磷酸二酯键、硫代磷酸酯键、酰胺键、胺键、硫醚键、醚键、酯键或碳-碳键实现；
    优选地，所述货物分子偶联至所述融合蛋白的N端或C端。
  13. 权利要求10所述的复合物，其中，所述融合蛋白与货物分子非共价连接。
  14. 权利要求13所述的复合物，其中，所述融合蛋白是权利要求9所述的融合蛋白，所述货物分子包含能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域；
    优选地，所述能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域是氨基酸序列；
    优选地，所述货物分子是肽或蛋白质；
    优选地，所述融合蛋白中的特异性结合序列包含亮氨酸拉链，所述货物分子包含所述亮氨酸拉链的反向序列，从而所述亮氨酸拉链能够与该反向序列形成异源二聚体；
    优选地，所述融合蛋白中的特异性结合序列包含亮氨酸拉链NZ(例如，SEQ ID NO:49所示的序列)，所述货物分子包含亮氨酸拉链CZ(例如，SEQ ID NO:50所示的序列)；
    优选地，所述融合蛋白中的特异性结合序列包含亮氨酸拉链CZ(例如，SEQ ID NO:50所示的序列)，所述货物分子包含亮氨酸拉链NZ(例如，SEQ ID NO:49所示的序列)。
  15. 权利要求13所述的复合物，其中，所述融合蛋白与货物分子通过静电作用缀合。
  16. 组合物，其包含权利要求1-9任一项所述的融合蛋白，以及货物分子；
    优选地，所述货物分子选自核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物；
    优选地，所述货物分子选自核酸、肽或蛋白；
    优选地，所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合。
  17. 权利要求16所述的组合物，其包含权利要求9所述的融合蛋白，所述货物分子包含能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域；
    优选地，所述能够与所述融合蛋白中的特异性结合序列发生特异性结合的结构域是氨基酸序列；
    优选地，所述货物分子是肽或蛋白质；
    优选地，所述融合蛋白中的特异性结合序列包含亮氨酸拉链，所述货物分子包含所述亮氨酸拉链的反向序列，从而所述亮氨酸拉链能够与该反向序列形成异源二聚体；
    优选地，所述融合蛋白中的特异性结合序列包含亮氨酸拉链NZ(例如，SEQ ID NO:49所示的序列)，所述货物分子包含亮氨酸拉链CZ(例如，SEQ ID NO:50所示的序列)；
    优选地，所述融合蛋白中的特异性结合序列包含亮氨酸拉链CZ(例如，SEQ ID NO:50所示的序列)，所述货物分子包含亮氨酸拉链NZ(例如，SEQ ID NO:49所示的序列)。
  18. 分离的核酸分子，其包含编码权利要求1-9任一项所述的融合蛋白、或权利要求11所述的复合物、或权利要求16或17所述的组合物的核苷酸序列。
  19. 载体，其包含权利要求18所述的分离的核酸分子。
  20. 宿主细胞，其包含权利要求18所述的分离的核酸分子或权利要求19所述的载体。
  21. 制备权利要求1-9任一项所述的融合蛋白、或者权利要求11所述的复合物的方法， 其包括，在合适的条件下培养权利要求20的宿主细胞，和从细胞培养物中回收所述融合蛋白或者复合物。
  22. 药物组合物，其包含权利要求1-9任一项所述的融合蛋白、权利要求10-15任一项所述的复合物、权利要求16或17所述的组合物、权利要求18所述的分离的核酸分子、权利要求19所述的载体或权利要求20所述的宿主细胞，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物包含权利要求10-15任一项所述的复合物，其中，所述货物分子是药学活性剂或可检测的标记。
  23. 权利要求1-9任一项所述的融合蛋白或包含编码所述融合蛋白的核苷酸序列的分离的核酸分子、载体或宿主细胞，用于制备药物的用途。
  24. 权利要求10-15任一项所述的复合物或权利要求16或17所述的组合物、或包含编码所述复合物或组合物的核苷酸序列的分离的核酸分子、载体或宿主细胞，在制备用于治疗疾病的药物中的用途；其中，所述复合物或组合物所包含的货物分子能够治疗所述疾病；
    优选地，所述疾病是与细胞程序性坏死相关的疾病，并且，所述货物分子包含蛋白磷酸酶1B；优选地，所述与细胞程序性坏死相关的疾病包括肝损伤(例如药源性肝损伤)、炎性疾病、缺血再灌注损伤和/或神经退行性疾病。
  25. 试剂盒，其包含权利要求1-9任一项所述的融合蛋白、权利要求10-15任一项所述的复合物、权利要求16或17所述的组合物、权利要求18所述的分离的核酸分子、权利要求19所述的载体或权利要求20所述的宿主细胞；
    优选地，所述试剂盒进一步包括用于转染和/或胞内递送的说明书。
  26. 权利要求1-9任一项所述的融合蛋白、权利要求1-15任一项所述的复合物、权利要求16或17所述的组合物、权利要求18所述的分离的核酸分子、权利要求19所述的载体或权利要求20所述的宿主细胞，作为递送试剂的用途。
  27. 用于将货物分子递送入细胞的方法，其包括使所述细胞与权利要求10-15任一项所述的复合物接触，其中所述复合物包括所述货物分子；
    优选地，所述细胞与所述复合物的接触在体外实施；
    优选地，所述分子选自核酸、肽或蛋白、糖类、脂质、化学化合物以及其任意的混合物；优选地，所述核酸选自DNA分子、RNA分子、siRNA、反义寡核苷酸、核酶、适体(aptamer)及其任意组合。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114716568A (zh) * 2022-03-18 2022-07-08 浙江农林大学 一种胰蛋白酶抑制剂修饰的β-酪蛋白纳米载体的构建方法及其应用
CN114716568B (zh) * 2022-03-18 2023-06-16 浙江农林大学 一种胰蛋白酶抑制剂修饰的β-酪蛋白纳米载体的构建方法及其应用

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