CN1634595A - Tie2 receptor mediated gene transfer system for targeted tumor gene therapy - Google Patents

Abstract

A gene tranfer system medicated by angiopoietin receptor Tie2 is provided. It includes (a) ligand oligopeptide combined specifically with Tie2 receptor, (b) poly cation polypeptide and any of (c) endosome released oligopeptide and (d) exogenous DNA. Said gene transfer system can effectively and targetedly import the exogenous gene to tumor new born vascular endotheliocyte and express the tumor cell of Tie2 receptor so as to suppress the tumor growth.

Description

Tie2 receptor-mediated gene transfer system for targeted tumor gene therapy

Technical Field

The present invention relates to the fields of molecular biology and gene therapy. In particular, it relates to a gene transfer system based on a ligand oligopeptide which binds to the angiopoietin receptor Tie 2. The transfer system guides exogenous DNA into a tumor neovascular endothelial cell and a tumor cell expressing a Tie2 receptor in a targeted way through receptor-mediated endocytosis, thereby achieving the purpose of treating tumors.

Background

PCT application PCT/CN97/00106(WO98/18951) discloses a novel receptor-mediated gene transfer system for targeted tumor gene therapy, wherein a gene transfer system mediated by three receptors, namely EGFR, IGF I/II R and VEGFR, is disclosed for tumor therapy. However, since there are many receptors on the surface of different tumor cells, there are many and few receptors expressed, and even some receptors are not expressed at all, so that only three systems cannot solve all the problems.

Partanen J et al discovered a new receptor type tyrosine kinase on the surface of human endothelial cells in 1992, named Tie2(Tyrosie kinase with Ig and EGF homology domains). The protein is a transmembrane protein consisting of 1124 amino acid residues. The receptor is composed of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region consists of two immunoglobulin-like domains, three EGF homology motifs and three fibronectin type III repeats; the intracellular region has a catalytic domain. Wherein the immunoglobulin-like domain is a binding site for a ligand.

Davis et al discovered a novel protein, Angiopoietin-1(Ang-1), in 1996, which is a ligand for the Tie2 receptor in the protein tyrosine kinase receptor family, with the Tie2 receptor being expressed almost exclusively on endothelial cells. Ang-1, structurally unlike known ligands for angiogenic factors or other protein tyrosine kinase receptors, does not directly promote the growth of cultured endothelial cells in vitro, so it is speculated that it may not be involved in the initiation of tumor neovasculature, but rather acts late in the angiogenic process to stabilize the vascular network.

After Ang-1 was discovered, by homologous screening, Maisonpierre discovered Angiopoietin-2(Ang2), which also binds to Tie2 receptor. In vitro Ang2 could not activate Tie2 receptors on endothelial cells, but could activate Tie2 receptors on other cells, such as NIH-3T3 cells, so that Ang2 might function as an antagonist to Ang1, leaving the vascular network in an unstable state, facilitating endothelial cell division and vascular network re-establishment.

The protein structure of these ligands was found to consist of three parts: a hydrophobic secretion signal peptide, a coded-Coil domain at the N-terminus and a fibrinogen-like domain at the C-terminus. The N-terminal conjugated-Coil domain is involved in multimerization thereof, while the C-terminal fibrinogen-like domain mediates binding of a ligand to a receptor. Expression of Tie2 is relatively specific, primarily on the surface of vascular endothelial cells, some tumor cells, and some hematopoietic cells. When ligand binds to receptor, receptor dimerization leads to Tie2 autophosphorylation, which can bind to multiple intracellular target proteins and exert different biological functions through different signaling pathways. Can resist endothelial cell apoptosis and promote endothelial cell migration by increasing phosphatidylinositol 3 kinase activity. The Tie2 knockout mice do not survive after birth, and pathological examination shows that the mice have blood vessels formed in the bodies but cannot form a complex blood vessel network. Due to the increased expression of Tie2 receptor in blood vessels of tumor tissues of various tumors, such as brain glioma, lung cancer, bladder cancer, and the like. Therefore, by blocking the signal conduction of Tie2 receptor, the tumor angiogenesis and the growth of tumor cells can be inhibited at the same time, and the purpose of treating tumors and diseases related to abnormal vascular proliferation is achieved.

The literature reports that the adenovirus is used for transducing the soluble Tie2 receptor extracellular region gene, the mouse breast cancer 4T1 and melanoma B16F10.9 cells can be inhibited in vivo, the inhibition rates respectively reach 64% and 47%, and the tumor metastasis is effectively inhibited. This study showed that inhibition of signal transduction at Tie2 receptor has some inhibitory effect on tumor growth.

In conclusion, Tie2 can be used as a new target for treating malignant tumors and some vascular abnormal proliferative diseases. The only molecules that can currently bind to Tie2 and have a certain function are the artificially prepared Tie2 antibody and the natural Tie2 ligand. Although natural ligands can be efficiently combined with Tie2 to achieve a good targeting effect, purified natural Tie2 ligands are complex in technology and unrealistic in large-scale preparation, and therefore cannot meet clinical application. Currently, only a small amount of human Tie2 ligand is used for scientific experiments and is expensive. In addition, the artificially prepared anti-Tie 2 antibody is a non-humanized macromolecular protein, is a foreign protein antigen molecule for human bodies, has strong immunogenicity, and is difficult to use clinically.

Therefore, there is an urgent need in the art to develop new Tie 2-targeted ligand oligopeptides and gene transfer lines for targeted tumor gene therapy based on Tie 2-ligand oligopeptides. The aim of treating diseases is achieved by gene therapy and other targeted therapeutic measures of targeted Tie 2.

Disclosure of Invention

The invention aims to provide a gene transfer system for Tie2 receptor-mediated targeted tumor gene therapy, which is constructed by ligand oligopeptides bound with Tie2 receptors and endocytosome release oligopeptides.

In a first aspect of the invention, there is provided a gene transfer system for growth factor receptor Tie2 mediated targeted gene therapy comprising (a) a ligand oligopeptide that specifically binds to the Tie2 receptor, wherein said ligand oligopeptide comprises the amino acid sequence of SEQ ID NO: 1.2 or 3; and (b) a polycationic polypeptide.

Preferably, the gene transfer system further comprises (c) an endocytosome-releasing oligopeptide.

In another preferred embodiment, the gene transfer system further comprises (d) an exogenous DNA.

In another preferred embodiment, the amino acid sequence of the ligand oligopeptide is SEQ ID NO: 1.2 or 3.

In another preferred embodiment, the polycationic polypeptide is selected from the group consisting of: polylysine, Polyacetimide (PEI), protamine, histone, adenovirus pV protein, adenovirus pVII protein, mu protein, proteins modified with polyethylene glycol (PEG) and mixtures thereof.

In another preferred embodiment, the exogenous DNA is selected from the group consisting of: eukaryotic recombinant expression vector DNA of anti-cancer genes, apoptosis genes, cytokine genes, oncogene antisense sequences, and mixtures thereof. More preferably, the exogenous DNA is selected from the group consisting of:

(i) an anti-cancer gene: p53, Rb;

(ii) apoptosis gene: caspase, p15, p16 and p21^WAF-1；

(iii) The cytokine genes comprise GM-CSF (granulocyte macrophage-colony stimulating factor) gene, TNF (tumor necrosis factor) α gene, INF (interferon) α, gamma gene, IL (interleukin) 2, IL3, IL4, IL12, IL15 and IL18 genes;

(iv) antisense sequence of oncogene: antisense sequences of oncogenes ras, c-myc, Akt;

(v) eukaryotic recombinant expression vectors: viral eukaryotic recombinant expression vectors (retrovirus recombinant expression vectors, adenovirus recombinant expression vectors and adeno-associated virus recombinant expression vectors) and non-virus eukaryotic recombinant expression vectors taking SV40 and CMV promoters as cis-regulatory elements.

In another preferred embodiment, two or three of the component (a) the ligand oligopeptide, (b) the polycationic polypeptide and (c) the endocytosome release oligopeptide are present as fusion proteins.

In another preferred embodiment, said endocytic corpuscle release oligopeptide element is selected from the group consisting of: HA20 or VP-1.

In a second aspect of the invention, there is provided an oligopeptide (less than 100, more preferably 50 amino acids in length) which binds to the Tie2 receptor and comprises the amino acid sequence: SEQ ID NO: 1.2 or 3.

In a third aspect of the invention, there is provided a method of preparing a gene transfer system for growth factor receptor Tie2 mediated targeted gene therapy comprising the steps of: mixing together (a) a ligand oligopeptide that specifically binds to Tie2 receptor, (b) a polycationic polypeptide, and optionally (c) an endocytosome-releasing oligopeptide to form a mixture. Preferably, for components (a), (b), (c), any two may be used to form a binary complex.

In a fourth aspect of the present invention, there is provided an antibody to the above ligand oligopeptide of the present invention.

In a fifth aspect of the invention, there is provided a conjugate comprising a ligand oligopeptide according to the invention as described above and a radionuclide, a chemical drug and a biotoxin coupled to said ligand oligopeptide. The conjugates can be directly applied to treat malignant tumors related to Tie2 overexpression, such as lung adenocarcinoma and osteosarcoma.

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprisinga safe and effective amount of a ligand oligopeptide of the invention, together with a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutical composition of the invention can be used for treating malignant tumors related to Tie2 overexpression, such as lung adenocarcinoma and osteosarcoma.

Drawings

FIG. 1 is a mass spectrum analysis chart of polypeptide GA 3.

FIG. 2 is¹²⁵The results of the test for specific binding of marker I GA3 to the stably expressed Tie2 SMMC7721 cell surface Tie2 receptor are shown schematically.

FIG. 3 is¹²⁵The specific binding test results of the I-labeled GA3 and rhTie2-Fc fusion protein are shown in a schematic diagram.

FIG. 4 is a drawing of¹²⁵The dissociation constant curve of the specific binding of the I-labeled GA3 and the Tie2 receptor has a Kd of 2.5X 10^-8M。

FIG. 5 shows the ELISA results of phage clones specifically binding to cell surface Tie2 receptor.

FIG. 6 shows the immunohistochemistry results of phage clones specifically binding to cell surface Tie2 receptor. FIG. A, B is the results of immunohistochemistry showing phage clones of GA4 and GA5, and Tie2(+) SMMC7721 cells, respectively; panels C and D are the immunohistochemistry results for phage clones displaying GA4 and GA5, respectively, bound to Tie2(-) SMMC7721 cells.

FIG. 7 is a diagram showing the results of the specific binding assay of phage clones to rhTie2-Fc fusion protein.

Detailed Description

The present inventors havemade extensive and intensive studies to prepare a ligand oligopeptide that specifically binds to Tie2 receptor and a gene transfer system based on the ligand oligopeptide. GA3(SEQ ID NO: 1) was obtained by amino acid sequence analysis of angiopoietins 1 and 2 using bioinformatics methods. GA4 and GA5(SEQ ID NO: 2 and SEQ ID NO: 3) are polypeptides that bind to Tie2 receptor molecules directly from peptide libraries by phage peptide library screening techniques. The specific position of the specific amino acid of the polypeptide is the active site of the polypeptide ligand binding with Tie2 receptor, namely the Tie2 receptor molecule polypeptide ligand motif, and determines the active characteristic of the polypeptide binding with Tie 2.

By utilizing the characteristic of the ligand oligopeptide for identifying the Tie2 receptor, exogenous DNA is selectively introduced into tumor neovascular endothelial cells or tumor cells expressing the Tie2 receptor through endocytosis of the cells, so that angiogenesis and tumor cells can be inhibited at the same time, and the aim of treating tumors is fulfilled. The present invention has been completed based on this finding.

Ligand oligopeptides

Ligand oligopeptides are key components of the targeting non-viral vectors of the invention. As used herein, "Ligand Oligopeptide (LOP)" and "receptor recognition oligopeptide" are used interchangeably to refer to any oligopeptide that recognizes and binds to target cells containing Tie2 receptors on their surface. As described above, the ligand oligopeptides of the present invention can specifically recognize and bind to Tie2 receptor. Specifically, the ligand oligopeptide of the invention refers to a polypeptide combined with the extracellular region of a cell membrane surface protein receptor Tie2, or a polypeptide combined with a soluble Tie2 molecule, and comprises a fusion polypeptide and an isolated polypeptide.

Ligand polypeptides of the invention also include variants of the sequence of SEQ ID NO.1-3 that have the same function of binding to Tie2 receptor. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus.

Those skilled in the art will recognize that non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, tyrosine, asparagine, and glutamine. Basic amino acids include lysine, histidine and arginine. Acidic amino acids include glutamic acid and aspartic acid. In the art, substitutions with amino acids having similar or analogous properties will not generally alter the function of the polypeptide. Furthermore, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein.

As used herein, the term "ligand oligopeptide" includes active fragments and active derivatives of GA3, GA4 and GA5 that bind to Tie 2.

As used herein, the term "ligand oligopeptide" also includes polypeptides in the form of amides or esters or salts thereof. Including both free, fused and chimeric polypeptides and polymers in which the polypeptide is monomeric. For example, a polypeptide comprising SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, a protein molecule, a chimeric protein molecule or a polymer of one, two or three amino acid sequences of the above-mentioned group.

In the specification and drawings of the present application, abbreviations used to represent bases, amino acids are recommended by the IUPCA-IUB Commission on Biochemical nomenclature. Wherein the amino acids are generally in the L form unless otherwise indicated. The polypeptide described in the present specification is, by convention, amino-terminated (N-terminal) at the left end and carboxy-terminated (C-terminal) at the right end. Generally when the C-terminus is a carboxyl group (-COOH) or a carboxylate (-COO-), it may be in the form of an amide (-COONH2) or an ester (-COOR). The ester residue R includes C1-6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, etc.), C3-8 cycloalkyl (e.g., cyclopentyl, cyclohexyl, etc.), C6-12 aryl (e.g., phenyl, a-naphthyl, etc.), C7-14 alkyl (e.g., benzyl, phenethyl, etc.).

One particularly preferred class of ligand oligopeptides are oligopeptides of 15 to 30 amino acids in length and containing a base unit sequence selected from the group consisting of:

WTIIQRREDG SVDFQRTWKE YK(SEQ ID NO：1)；

HATGTHGLSL SH(SEQ ID NO：2)；

NSLSNASQFR AP(SEQ ID NO：3)。

particularly preferred examples are the following oligopeptides:

oligopeptide GA3, the amino acid sequence of which is WTIIQRREDG SVDFQRTWKE YK (SEQ ID NO: 1);

oligopeptide GA4, whose amino acid sequence is HATGTHGLSL SH (SEQ ID NO: 2);

oligopeptide GA5, whose amino acid sequence is NSLSNASQFR AP (SEQ ID NO: 3);

the amino acid sequences of GA3, GA4, and GA5 were obtained from bioinformatic analysis of the amino acid sequences of angiopoietins 1 and 2.

In addition, the ligand oligopeptide of the present invention also includes one or more (usually 1-8, preferably 1-5) sites of amino acids, and the formed ligand oligopeptide can be replaced by amino acids with similar properties.

The ligand oligopeptides of the present invention may contain a single base unit sequence, or multiple (e.g., 2-10) base unit sequences.

The ligand oligopeptide of the present invention can be conveniently prepared by artificial synthesis or genetic engineering recombination. For the preparation of these synthetic oligopeptides, reference is made to various publications such as WO98/18951(PCT/CN 97/00106).

The invention also provides coding sequences of Tie2 ligand oligopeptides such as GA3, GA4, GA5 and the like, which respectively contain the amino acid sequences shown in SEQ ID NO: 5. 6 and 7. These coding sequences can be obtained by conventional artificial synthesis methods or recombinant methods.

Polycationic polypeptides

Polycationic polypeptides are another key component of the targeted non-viral vectors of the present invention.

As used herein, "polycationic polypeptide" refers to a class of polypeptides rich in basic amino acids, which have a basic amino acid (lysine, histidine and arginine) ratio of about 20% or more, and thus are positively charged, and can bind to negatively charged DNA by electrostatic attraction, making the DNA more stable.

The polycationic polypeptide usable in the present invention is not particularly limited as long as it has a basic amino acid (lysine and arginine) ratio of about 20% or more, preferably 30% or more, more preferably 40% or more, and thus is positively charged. Representative examples include: polylysine, polyacetimide, protamine, histone, adenovirus pV protein, adenovirus pVII protein, m μ protein, the above proteins modified by polyethylene glycol (PEG), and mixtures thereof.

Endocytic corpuscle release oligopeptide element

The targeting non-viral vector of the invention can also contain an endocytosome release oligopeptide element which plays a role in preventing DNA endocytosed into cells from being degraded.

Any endocytosome-releasing oligopeptide may be used in the present invention, representative examples include (but are not limited to): HA20, VP-1.

Targeted non-viral vectors

As used herein, a "targeting nonviral vector" of the present invention refers to a mixture or complex comprising the following components (a) and (b), or comprising components (a), (b), and (c): (a) a ligand oligopeptide that specifically binds to Tie2 receptor, (b) a polycationic polypeptide, (c) an endocytosome-releasing oligopeptide.

In the present invention, the components (a), (b) and (c) may be in a non-linked form, or any two or three of them may be linked together by a covalent or non-covalent means. Particularly preferred forms include: the composition comprises a ligand oligopeptide-polycation polypeptide binary compound, a polycation polypeptide-endocytosis corpuscle release oligopeptide binary compound and a ligand oligopeptide-polycation polypeptide-endocytosis corpuscle release oligopeptide ternary compound. For example HA 20-polylysine, HA 20-protamine, HA 20-histone covalent links, etc.

The linkage may be by chemical means (e.g.using a coupling agent such as SPDP) or by recombinant means, for example to form a fusion protein.

For fusion proteins, endocytosis is reduced in (a) a ligand oligopeptide, (b) a polycationic polypeptide, and (c)The body release oligopeptide elements can optionally contain connecting peptides between the body release oligopeptide elements so as to increase the flexibility and the spreadability of the spatial structure of the composite polypeptide and enable the elements to play respective functions. The linkerpeptide usable in the present invention is not particularly limited as long as it functions as a linker. An example of a class of linker peptides is (Gly)_2-6Ser, e.g. (Gly)₄And (6) Ser. The linker peptide may be used in a plurality of tandem.

The (a) ligand oligopeptide, (b) polycationic polypeptide, and (c) endocytosome release oligopeptide element of the present invention can be replaced with amino acids of similar properties, as long as the respective similar biological functions are retained.

Preparation method of targeting non-viral vector

Generally, the targeting nonviral vector of the present invention can be obtained by mixing the above-mentioned (a), (b) and (c) components or a complex thereof. Wherein the mixing ratio of the components (a), (b) and (c) is usually 0.5-1: 1-2: 0-1, preferably 0.8-1: 1-1.5: 0.5-1.

Further, when administered in the form of (a) - (b) complex and (b) - (c) complex, (a) - (b) complex: (b) the mixing ratio of the compound (c) to the compound (c) is usually 0.8-1.2: 0.8-1.2, preferably 0.9-1.1: 0.9: 1.1.

Exogenous DNA

The foreign DNA usable in the present invention is not particularly limited, and may be various therapeutic or prophylactic DNAs, such as a target gene, an anti-sense oncogene, an anticancer gene, a suicide gene, an apoptosis gene, a cytokine gene, or a combination thereof, or eukaryotic expression vector DNAs containing the above genes. The protooncogene antisense sequence includes protooncogene (ras)^H、ras^K、ras^NC-myc, bcl-2, Akt), antisense DNA sequences, antisense oligonucleotides and antisense oligodeoxynucleotides of growth factors and their receptor genes; the cancer suppressor gene comprises p53, Rb and PTEN, and the suicide gene comprises HSV-TK (herpes simplex virus thymidine kinase) gene and CD (escherichia coli cytosine deaminase) gene; the apoptosis genes include p15, p16, p21^WAF-1The cell factor gene includes GM-CSF (granulocyte macrophage colony stimulating factor) gene, TNF α (tumor necrosis factor) gene, INF α, gamma gene, IL (interleukin) 2, 3, 4, 12, 15, 18 gene, etc.

Targeted gene transfer system

The targeted non-viral vector of the invention is mixed with exogenous DNA to form a gene transfer system of Tie2 receptor mediated targeted tumor gene therapy. Depending on the foreign DNA used, the gene transfer system of the present invention can be used for the treatment of genetic diseases or diseases such as tumors, particularly for gene therapy.

Pharmaceutical composition

The invention also provides a pharmaceutical composition, which contains a safe and effective amount of one or more targeting non-viral vectors of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.

When administered, the pharmaceutical composition further comprises one or more of the above-described exogenous DNA.

The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The main advantage of the present invention is that,

(1) based on the mechanism of protein molecule interaction, the invention uses small molecule polypeptide to simulate the binding site of natural Tie2 ligand and Tie2 to bind Tie 2. Compared with monoclonal antibodies, the small molecular polypeptide has a clear structure, is limited by a binding site with Tie2, is not easy to generate steric hindrance effect between polypeptides, and has no interference of Fc segments.

(2) The small molecular polypeptide has the characteristics of almost no antigenicity, small side effect, easy preparation, large-scale production, convenient use and the like.

(3) The polypeptide of the present invention can be combined with cell surface Tie2 receptor protein to regulate angiogenesis via activating or blocking Tie2 signal transduction path, so that it may be used as precursor or medicine composition for clinical medicine development, and includes DNA encoding the polypeptide of the present invention and antibody of the polypeptide of the present invention.

(4) The related polypeptide can be used as a targeting peptide for a gene transfer system to perform targeted gene therapy aiming at tumor neovascular endothelial cells and tumor cells with high expression of Tie2 receptors.

(5) The related polypeptide can be used as a targeting peptide to be connected with radionuclide, chemical drugs and biotoxin and has the targeting treatment effect on malignant tumors and Tie2 system function disorder diseases.

(6) The polypeptide and the DNA sequence for coding the polypeptide can also be used as a detection or diagnostic reagent.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

Example 1 chemically synthesized polypeptide and Mass Spectrometry analysis thereof

In this example, GA3, GA4, and GA5 were prepared by a synthetic method.

The polypeptide was identified as polypeptide GA3[ SEQ ID NO: for example, the synthesis is carried out on a polypeptide synthesizer by a solid phase synthesis method. The carrier resin used was an HMP amino resin with an amino acid amount of 1 millimole, i.e. 4: 1 amino acid to resin. According to polypeptide GA3[ SEQ ID NO: 1]amino acid sequence, and sequentially adding each amino acid component for solid phase synthesis. After the polypeptide is synthesized, cutting according to the steps recommended by PE company, filtering the reacted mixed solution by using a G4 glass sand funnel to remove resin, evaporating the filtrate to 1ml at normal temperature under low pressure, adding 50ml of precooled ether to precipitate the polypeptide, standing overnight at 4 ℃, filtering by using a G6 glass sand funnel, and performing vacuum freeze drying to obtain a crude polypeptide product. Purifying with Sephadex G10 column, eluting with 0.1N glacial acetic acid, collecting the first peak, concentrating, removing acid, dissolving with small amount of water, and vacuum freeze drying to obtain dry powder. Mass spectrometry analysis is carried out, the molecular weight of the obtained polypeptide is similar to the theoretical value, and is 2842, which shows that the polypeptide is GA3[ SEQ ID NO: 1](fig. 1).

The molecular weight of GA4 obtained by the same method is 1380, which is close to theoretical value, and shows that the polypeptide is GA4[ SEQ ID NO: 2].

GA5 produced by the same method, with a measured molecular weight of 1456, was close to theoretical, indicating that this polypeptide is exactly GA5[ SEQ ID NO: 3].

Example 2 GA3 binding assay specific for Tie2 receptor on the surface of SMMC7721 cells stably expressing Tie2

(one) establishment of cell line stably expressing Tie2 extracellular region

(1) A human cell strain SMMC7721 which is negatively expressed by Tie2 is screened by a conventional Western blotting method, and the method comprises the following steps:

collecting different tumor cell strains with good growth on a plate (9cm), washing with PBS for three times, adding 1ml of PBS, scraping the cells with a curette, collecting into a 1.5ml centrifuge tube, 8000rpm for 1min, removing the supernatant, adding 100 μ l of protein lysate (containing 1 μ g of protease inhibitor), placing on ice for lysis for 30 min, 10000rpm, centrifuging for 10min, and transferring the supernatant into a new tube to obtain the total cell protein. 20 mul of protein solution is respectively added with 4 mul of sample adding buffer solution, boiled for 10min, at 10000rpm, centrifuged for 10min, supernatant is taken for protein electrophoresis, membrane is transferred, 0.25 percent skim milk/PBS is sealed, 1: 2000 Tie2 extracellular region antibody (Santa Cruz biotechnology) is added, the mixture is kept overnight at 4 ℃, PBS is washed for three times, 5 minutes/time, 1: 5000 secondary antibody (Santa Cruz biotechnology), PBS is washed for three times, 5 minutes/time, ECL reacts for 5 minutes, and tabletting, developing and fixing are carried out. The results showed that the SMMC7721 cell line was negative in expression.

(2) Construction of PCDNA3.0-ExTie2 plasmid

An upstream primer: 5'-CCG GAA TTC ATG GAC TCT TTA GCC AGC TT-3' (SEQ ID NO: 8)

A downstream primer: 5'-CCG CTC GAG CTA GAT GGC TAT AAG CAG CAT-3' (SEQ ID NO: 9)

The EcoRI and XhoI restriction site-introduced oligonucleotide pair is used as a primer, pAdvtie2 plasmid (purchased from the institute of tumor research in Shanghai) is used as a template, a DNA fragment for coding a Tie2 extracellular region (ExTie2) is obtained through PCR, EcoRI and XhoI double-restriction reaction is carried out on a PCR product and PCDNA3.0 carrier plasmid (purchased from Invitrogen company), gel electrophoresis is carried out, the carrier fragment and the insert fragment are recovered through gel cutting, the carrier fragment and the insert fragment after the restriction are connected through T4 ligase, and the plasmid is transformed into competent Escherichia coli cells (DH5 α) (purchased from Invitrogen company) overnight at 16 ℃, plated (Amp resistance), cultured overnight at 37 ℃, single-clone amplified, and the plasmid is extracted in a small amount, identified through restriction and sequenced to obtain the PCDNA3.0-ExTie2 plasmid.

(3) Introducing PCDNA3.0-ExTie2 plasmid into the SMMC7721 cells by a liposome transfection method, screening by G418, selecting single clone, amplifying, extracting total protein, and identifying by Western blot to obtain an ExTie2 stable expression cell line.

(II),¹²⁵Specific binding of I GA3 to Tie2 receptor on SMMC7721 cell surface stably expressing Tie2

Inoculation of SMMC7721 cells stably expressing Tie2 in 96-well plates at 5X 10³Cells/well, cultured overnight for cell attachment, washed 3 times with PBST (PBS + 0.1% Tween20), 20. mu.l of pre-cooled GA3 (containing 320pmol of GA) diluted with binding buffer PBS +20mM HEPES +2.5mg/ml BSA, pH7.4) added to the competitive group, 20. mu.l of binding buffer added to the non-competitive group, with three wells in parallel per group; at the same time, the iodine label GA3 was diluted to 1ml with binding buffer, and 20. mu.l (contained) was added to each well¹²⁵I ga30.02pmol). Binding was carried out at 4 ℃ for 3 hours. PBST was washed 3 times, 100. mu.l of 0.25% pancreatin was added, digested for 10 minutes, transferred to assay tubes, washed with 100. mu.l of PBST, the same wash solution was added to the corresponding assay tubes, and gamma counted.

The results of the experiment are shown in FIG. 2. It shows that GA3 can be specifically combined with the Tie2 receptor on the surface of SMMC7721 cells which stably express Tie 2.

Example 3¹²⁵Specific binding of I-tag GA3 and rhTie2-Fc fusion protein

0.1M NaHCO per well₃(pH8.6) 50. mu.l of coated 1. mu.g of rhTie2-Fc fusion protein (purchased from R)&Company D) in 96-well plates, overnight at 4 ℃, PBST washed 3 times, the competitor group added pre-cooled GA3 diluted with binding buffer (PBS +20mM HEPES +2.5mg/ml BSA pH7.4) in 20. mu.l (containing 320pmol of GA) per well, the non-competitor group added 20. mu.l binding buffer, each group in parallel with three wells; at the same time, the iodine label GA3 was diluted to 1ml with binding buffer, and 20. mu.l (contained) was added to each well¹²⁵I ga30.02pmol). Binding was carried out for 1 hour at room temperature. PBST was washed 3 times, eluted with 100. mu.l of Glysine-HCl (pH2.2) for 15 minutes, transferred to assay tubes, washed with 100. mu.l of Glysine-HCl, added to the corresponding assay tubes, and gamma-counted.

The results are shown in FIG. 3. It shows that GA3 can be specifically combined with rhTie2-Fc fusion protein.

Example 4 GA3 affinity assay for Tie2 receptor

Inoculation ofSMMC7721 cells stably expressing Tie2 in 96-well plates at 1X 10⁴Cells/well, incubated overnight until cells were adherent, washed 3 times with PBST (PBS + 0.1% Tween20), blocked for 1 hour at 4 ℃ with 100. mu.l blocking buffer (blocking buffer) (0.05mM PB +5mg/ml BSA), washed 3 times with PBST, and added to each well according to the following concentration gradient¹²⁵I-labelled GA3, three parallel wells were set for each concentration set, and the reaction system was 100. mu.l per well. Binding for 3 hours at 4 ℃ and PBST washing 3 times, adding 100. mu.l of 0.25% pancreatin, digesting for 10 minutes, transferring to the assay tube, washing with 100. mu.l PBST, adding the same wash to the corresponding assay tube, and gamma counting. From the results of the experiments, the dissociation constant Kd [ concentration gradient: 6.13nM, 12.5nM, 25nM, 50nM, 100nM, 200nM, 300nM, 500nM, 750nM]。

The results are shown in FIG. 4. The Kd of GA3 binding to Tie2 receptor was determined to be 2.5X 10^-8M, which is much higher than the currently existing Tie2 ligand oligopeptide.

Example 5 phage clones specifically bind to cell surface Tie2 receptor

(1) ELISA assay

The SMMC7721 cells of Tie2(+) and Tie2(-) in logarithmic growth phase were inoculated into 96-well plates at 1X 10⁴Culturing cells/well overnight for cell attachment, washing with PBS 3 times, coating rhTie2, BSA and skimmed milk onto 96-well plate (500ng/ul), blocking with BSA, washing with PBS 3 times, adding 1 × 10 per well¹²pfu amplified and purified phage, 37 degrees 1 hours. After PBST washing 6 times, HRP-Mu.l/well of goat anti-M13 polyclonal antibody was added at 37 ℃ for 1 hour. After PBST rinsing, an enzyme reaction substrate (ABST) solution was added for color development, and OD405nm was detected by an enzyme-linked immunosorbent assay. The experiment set up a blank including cell-free wells for each clone and wild phage without insert. Each set was set with 3 parallel wells.

The ELISA results are shown in FIG. 5. Phage displaying GA5 were shown to bind to rh-Tie2 and SMMC7721 cells stably expressing Tie 2.

(2) Immunohistochemical analysis

(a) Preparation of phage displaying GA4 and GA5

Preparation of phage displaying GA 4:

separately with 0.1M NaHCO₃(pH8.6) 50. mu.l of a coating solution of 5. mu.g of recombinant human Tie2(rh-Tie2) were placed in a 96-well plate wet box overnight at 4 ℃. PBST 6 washes and blocking buffer (0.1M NaHCO)₃(pH8.6) 100. mu.l +5mg/ml BSA pH7.5) blocking at 4 ℃ for 3 hours. TBST was washed 10 times, and 10. mu.l (4X 10) of the resulting solution was taken out¹⁰pffudge) original library phage (New England BioLabs Inc.), diluted with 1ml TBST and added to the cell culture plate, and destained and shaken gently on a shaker for 1 hour. The unbound phage were removed by inverting on clean filter paper. TBST was washed 10 times, 100. mu.l of Glysine-HCl (pH2.2) was added for elution for 8 minutes, the eluate was transferred to a measuring tube, 15. mu.l of Tris-HCl (pH9.1) was added for neutralization, 1. mu.l of the eluate containing phages was titrated, and the remainder was added to ER2537 for amplification. And repeating the previous steps to carry out second, third and fourth screening. Newly recovered ER2537 single colonies were picked, inoculated in 2ml LB, expanded to mid-log phase for use. Mu.l of the fourth eluate was titrated, a single blue colony was picked up and added to 2ml of LB together with 20. mu.l of middle log-stage ER2537 cells, and shaken in a shaker at 37 ℃ for 4.5 hours. Collecting the bacterial liquid in a 1.5ml centrifuge tube, centrifuging at 4 ℃ and 10000rpm for 10min, carefullytaking 500 mul of supernatant to a new tube (taking a small amount to be stored at 4 ℃ for subsequent amplification), adding 200 mul of PEG/NaCl, mixing uniformly, standing at room temperature for 10min, centrifuging at 10000rpm for 10min, removing the supernatant, re-centrifuging, and carefully removing the supernatant. Add 100. mu.l lodide buffer and mix well, add 250. mu.l ethanol at the same time, stand 10min at room temperature, centrifuge 10min at 10000rpm, remove supernatant, wash with 70% ethanol once, precipitate is dissolved in 30. mu.l TE for sequencing. Sequencing revealed that the phage displaying GA4 appeared significantly enriched. Mu.l of the phage supernatant containing the display GA4, which had been preserved, was added to 20ml of LB together with 200. mu.l of metaphase ER2537, and shaken on a shaker at 37 ℃ for 4.5 hours. Collecting bacterial liquid to a centrifuge tube, centrifuging at 4 ℃ and 10000rpm for 10min, transferring supernatant to a new tube, re-centrifuging, centrifuging at 10000rpm for 10min, taking 80% of supernatant to the new tube, adding 1/6 volumes of PEG/NaCl, and standing overnight at 4 ℃. Centrifuging at 10000rpm for 15min, removing supernatant, re-centrifuging, and removing residual supernatant. Resuspending 1ml TBS, transferring to 1.5ml centrifuge tube, centrifuging at 10000rpm for 10min, takingPlacing 80% of the clear solution into a new tube, adding 1/6 volumes of PEG/NaCl, and carrying out ice bath for 1 hour;centrifugation was carried out at 10000rpm for 10 minutes, the supernatant was removed, and 200. mu.l TBS/0.02% NaN was used₃Resuspend, centrifuge at 10000rpm for 1min, transfer supernatant into new tube, i.e. amplified phage displaying GA 4.

Phage displaying GA5 were prepared at 1X 10⁴SMMC7721-ExTie2 cells were used as the target for selection and eluted with TBS containing Ang-2 (100. mu.g/ml). The remaining steps were the same as for the preparation of the GA 4-displayingphage.

(b) Binding assays

Tie2(+) and Tie2(-) SMMC7721 cells were seeded in 96-well plates at 1X 10⁴Culturing overnight for cell adherence, washing with PBST for 3 times, fixing with 10% formaldehyde at 37 deg.C for 30 min, washing with PBST for 3 times, 5 min/time, adding 0.3% hydrogen peroxide (methanol) at 37 deg.C for 30 min, washing with PBST for 3 times, and adding 10¹¹The phages displaying GA4 and GA5 (containing 0.1% BSA) were incubated at 37 ℃ for 1 hour. PBST was washed 6 times, 100. mu.l/well of HRP-goat anti-M13 polyclonal antibody was added, and the mixture was incubated at 37 ℃ for 1 hour. And (3) adding DAB working solution to stop light for color development after PBST is rinsed, and stopping the reaction when the color development is proper.

The results are shown in FIG. 6. Panel A is a graph of immunohistochemistry for GA 4-displaying phages bound to Tie2(+) SMMC7721 cells, panel B is a graph of immunohistochemistry for GA 5-displaying phages bound to Tie2(+) SMMC7721 cells, panel C is a graph of immunohistochemistry for GA 4-displaying phages bound to Tie2(-) SMMC7721 cells, and panel D is a graph of immunohistochemistry for GA 5-displaying phages bound to Tie2(-) SMMC7721 cells.

The experimental results all confirm that the fusion polypeptides (GA4 and GA5) related to the invention can be specifically combined with the cell surface Tie2 receptor protein.

Example 6 phage cloning specific binding to rhTie2-Fc fusion protein

0.1M NaHCO per well₃(pH8.6) 50. mu.l of 1. mu.g of rhTie2-Fc fusion protein (R)&Company D) were washed 3 times in a 96-well plate overnight at 4 ℃ in PBST, blocked with blocking buffer (PBS +5mg/ml BSA pH7.5) for 3 hours at 4 ℃ while a negative control (coated with 1. mu.g/50. mu.l BSA) and a blank were set up. PBST washed 3times, added 2X 10¹⁰pfu bacteriophage(containing 0.1% BSA) was incubated at 37 ℃ for 1 hour. PBST was washed 6 times, eluted with 100. mu.l of Glysine-HCl (pH2.2) for 8 minutes, the eluate was transferred to a measuring tube, neutralized with 15. mu.l of Tris-HCl (pH9.1), and then the recovered phage was titrated to calculate binding efficiency.

The results of the experiment are shown in FIG. 7. It was shown that both GA 4-and GA 5-displaying phages were able to bind rh-Tie 2.

Example 7 construction of non-viral vectors

A. Obtaining the Components

(1) Synthesis of ligand oligopeptide GA:

the peptides GA3, GA4 and GA5 were obtained by column chromatography using a peptide synthesizer of ABI, USA, according to the manual of peptide synthesis, and the amino acid sequences thereof were as follows:

WTIIQRREDG SVDFQRTWKE YK(SEQ ID NO：1)；

HATGTHGLSL SH(SEQ ID NO：2)；

NSLSNASQFR AP(SEQ ID NO：3)。

(2) poly L-lysine (Poly-L-lysine, abbreviated as P.L.)

Purchased from SIGMA, USA, with a molecular weight distribution of 15000-30000 daltons.

(3)HA20

The oligopeptide HA20 is obtained by using a polypeptide synthesizer of American ABI company and synthesizing according to a polypeptide synthesis operation manual, and performing column chromatography separation, wherein the amino acid sequence of the oligopeptide HA20 is GLFEA IAEFI EGGWE ELIEG (SEQ ID NO: 4).

B. Preparation of binary complexes

First, GA (GA3 or GA4 or GA5), HA20 and Poly L-lysine (Poly-L-lysine, abbreviated as P.L) were dissolved in PSB (0.1M sodium phosphate buffer, pH7.4, containing 0.1M NaCl), and then, the reaction was carried out as follows.

(1)

Note: SPDP is N-succinimidyl-3- (2-pyridyldithio) -propionate.

The molar ratio of P.L. to SPDP (N-) was 1: 5, the reaction time was 2 hours and the temperature was 25 ℃. After the reaction was completed, unreacted SPDP was removed by dialysis. P.l. -PDP freeze-drying.

(2) P.L. -PDP dry powder dissolved in PSB

Note: DTT is dithiothreitol.

The DTT was used in excess for 1 hour at 25 ℃. After the reaction was completed, unreacted DTT was removed by dialysis. The product was freeze dried.

(3)

GA. The molar ratio of HA20 to SPDP was 1: 5, the reaction time was 2 hours and the temperature was 25 ℃. After the reaction, unreacted SPDP was removed by dialysis through a dialysis bag of MWCO 1000. The product was freeze dried.

(4) The GA-PDP, HA20-PDP and P.L. -SH dry powders were all dissolved in PSB.

The molar ratio of GA-PDP, HA20-PDP and P.L. -SH is 3: 1, the reaction time is 24 hours, and the temperature is 25 ℃. After completion of the reaction, unreacted GA-PDP and HA20-PDP were removed by dialysis.

C. Preparation of Targeted non-viral vectors

Mixing the P.L. -GA and the P.L. -HA20 according to the mass ratio of 1: 1, 0.8: 1.2 and 1.2: 0.8 respectively to obtain the required non-viral vector.

Alternatively, GA, polylysine and HA20 were mixed together in a mass ratio of 1: 1.5: 1 to form the desired non-viral vector.

EXAMPLE 8 extraction and purification of plasmid DNA

The isolation and purification of the recombinant eukaryotic expression vector plasmid DNA used in this example was a pure DNA product obtained using the Maxi plasmid kit from QIAGEN.

A single clone was selected from the LB plate and inoculated into 5ml of LB medium containing ampicillin, and shaken overnight at 37 ℃ and 250 rpm. The cells were transferred 1: 1000 into 200ml of LB medium containing ampicillin, and shaken at 37 ℃ and 250rpm for 16 hours. The transferred cell suspension was centrifuged at 4,000rpm at 4 ℃ for 10 minutes in a 500ml centrifuge tube, and the supernatant was discarded to collect cells. The pellet was resuspended in 10ml Buffer P1(50mM Tris. Cl, pH8.0, 10mM EDTA pH8.0, 100. mu.g/ml RNase A). After 10ml of Buffer P2(200mM NaOH, 1% SDS) was added, the mixture was gently inverted 4 to 6 times, mixed, and allowed to stand at room temperature for 5 minutes. 10ml of Buffer P3(3.0M NaAc, pH5.5) was added thereto, the mixture was mixed by inversion, and then transferred to a 50ml centrifuge tube, centrifuged at 10,000rpm at 4 ℃ for 30 minutes, and the supernatant was collected. The supernatant was filtered aseptically through four layers into a QIAGEN Maxi plasmid extraction column (equilibrated with 10ml Buffer QBT in advance), allowed to flow down naturally, after the supernatant was completely loaded into the column, 30ml Buffer QC was added and washed twice, and finally 15ml Buffer QF was added, the effluent was collected, 0.7 times volume of isopropanol was added, 10,000rpm was centrifuged at 4 ℃ for 30 minutes, the supernatant was discarded, 1 time of washing with 75% ethanol and precipitation, naturally dried at room temperature, dissolved in a suitable volume of TE (pH8.0), the DNA mass was confirmed by 1% agarose gel electrophoresis, and the DNA was confirmed by UV spectrophotometer for purity and quantification and stored at-20 ℃.

Example 9 construction of Quaternary Complex Gene transfer System

1) GA-p.l. and HA20-p.l. were dissolved in water, filtered through a 0.22 μm filter for sterilization, and stored at-20 ℃ for further use. All plasmids were extracted and purified by QIAGEN column, precipitated with 2 volumes of alcohol and 1/3 volumes of 3M NaAc, washed once with 75% alcohol, and dried in air. The resulting solution was dissolved in a suitable amount of MilliQ water to give a concentration of 0.2. mu.g/. mu.l.

2) The optimal mass ratio between the non-viral vector and the DNA when they form a complex.

0.2. mu.g of CMV- β -gal plasmid DNA and a non-viral vector were mixed at a mass ratio of 1: 0.5, 1: 1, 1: 1.5, 1: 2, left at 25 ℃ for 30 minutes, and the blocking condition of the DNA was identified by 1% agarose gel electrophoresis to determine the optimum mass ratio.

3) Preparing the quaternary complex gene transfer system according to the determined proportion.

Example 10 experiment of in vitro introduction of CMV- β -gal Gene transduced by GA Gene introduction System

1) Cell culture

SPC-A1 cell subculture: SPC-A1 cells were digested with 0.25% trypsin, inoculated into a cell culture flask, added with 10% calf serum-containing DMEM complete medium, and cultured at 37 deg.C with 5% CO₂Culturing in an incubator.

2) In vitro introduction experiment

The cultured cells were digested with 0.25% trypsin and then seeded in six-well plates at 1.5X 10/well⁵On the next day, when the cell fullness was about 60%, quaternary complex gene transfer systems (i.e., three quaternary complex gene transfer systems based on GA3, GA4, and GA5, respectively) equivalent to 2. mu.g of DNA were added to 1ml of the culture medium. After 24 hours of transfection, fresh DMEM complete medium containing 10% calf serum was replaced, and culture was continued for 24 hours. Then, the cells were washed 2 times with PBS, and then fixed for 10 minutes at room temperature by adding a freshly prepared fixative, rinsed 3 times with PBS, each for 15 minutes, to completely remove the fixative, then blotted dry with filter paper for PBS on the tumor mass and organs, and then incubated for 24 hours at 37 ℃ with a freshly prepared staining solution, and then the condition of exogenous gene introduction (staining) was observed (staining was performed)The liquid composition is as follows: 1mg/ml X-gal, 5mM K₄[Fe(CN)₆]，5mMK₃[Fe(CN)₆]，2mM MgCl₂)。

As a result, β -gal gene-transduced cells of the present invention were blue, and the control group containing physiological saline and plasmid alone had no exogenous gene introduced.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by theappended claims.

Sequence listing

<110>Shanghai New world Gene technology development Co., Ltd

<120>Tie2 receptor mediated gene transfer system for targeted tumor gene therapy

<130>034757

<160>9

<170>PatentIn version 3.1

<210>1

<211>22

<212>PRT

<213>Artificial sequence

<220>

<221>MISC_FEATURE

<223>ligand oligopeptide GA3 binding to Tie2 receptor

<400>1

Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln Arg

1 5 10 15

Thr Trp Lys Glu Tyr Lys

20

<210>2

<211>12

<212>PRT

<213>Artificial sequence

<220>

<221>misc_feature

<223>ligand oligopeptide GA4 binding to Tie2 receptor

<400>2

His Ala Thr Gly Thr His Gly Leu Ser Leu Ser His

1 5 10

<210>3

<211>12

<212>PRT

<213>Artificial sequence

<220>

<221>misc_feature

<223>ligand oligopeptide GA5 binding to Tie2 receptor

<400>3

Asn Ser Leu Ser Asn Ala Ser Gln Phe Arg Ala Pro

1 5 10

<210>4

<211>20

<212>PRT

<213>Artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(20)

<223>endocytic corpuscle releasing oligopeptide HA20

<400>4

Gly Leu Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu Glu

1 5 10 15

Leu Ile Glu Gly

20

<210>5

<211>66

<212>DNA

<213>Artificial sequence

<220>

<221>misc_feature

<223>coding sequence of ligand oligopeptide GA3

<400>5

tgtacaatta ttcagcgacg tgaggatggc agcgttgatt ttcagaggac ttggaaagaa 60

tataaa 66

<210>6

<211>36

<212>DNA

<213>Artificial sequence

<220>

<221>misc_feature

<223>coding sequence of ligand oligopeptide GA4

<400>6

catgctacgg gtactcatgg gctttcgctt tctcat 36

<210>7

<211>36

<212>DNA

<213>Artificial sequence

<220>

<221>misc_feature

<223>coding sequence of ligand oligopeptide GA5

<400>7

aattcgctgt cgaatgcttc ggagtttcgg gcgccg 36

<210>8

<211>29

<212>DNA

<213>Artificial sequence

<220>

<221>misc_feature

<223>primer

<400>8

ccggaattca tggactcttt agccagctt 29

<210>9

<211>30

<212>DNA

<213>Artificial sequence

<220>

<221>misc_feature

<223>primer

<400>9

ccgctcgagc tagatggcta taagcagcat 30

Claims (10)

1. A gene transfer system for growth factor receptor Tie2 mediated targeted gene therapy comprising (a) a ligand oligopeptide that specifically binds to the Tie2 receptor, wherein said ligand oligopeptide comprises the amino acid sequence of seq id NO: 1.2 or 3; and (b) a polycationic polypeptide.

2. The gene transfer system of claim 1, further comprising (c) an endocytosome-releasing oligopeptide.

3. The gene transfer system according to claim 1 or 2, further comprising (d) an exogenous DNA.

4. The gene transfer system of claim 1, wherein the ligand oligopeptide has the amino acid sequence of SEQ ID NO: 1.2 or 3.

5. The gene transfer system of claim 1, wherein the polycationic polypeptide is selected from the group consisting of: polylysine, polyacetimide, protamine, histone, adenovirus pV protein, adenovirus pVII protein, m μ protein, polyethylene glycol modified proteins and mixtures thereof.

6. The gene transfer system of claim 3, wherein the exogenous DNA is selected from the group consisting of: eukaryotic recombinant expression vector DNA of anti-cancer genes, apoptosis genes, cytokine genes, oncogene antisense sequences, and mixtures thereof.

7. The gene transfer system of claim 3, wherein the exogenous DNA is selected from the group consisting of:

(i) an anti-cancer gene: p53, Rb;

(ii) apoptosis gene: caspase, p15, p16 and p21^WAF-1；

(iii) The cytokine genes comprise GM-CSF gene, TNF α gene, INF α, gamma gene, IL2, IL3, IL4, IL12, IL15 and IL18 genes;

(iv) antisense sequence of oncogene: antisense sequences of oncogenes ras, c-myc, Akt;

(v) eukaryotic recombinant expression vectors: viral eukaryotic recombinant expression vector and non-viral eukaryotic recombinant expression vector with SV40 and CMV promoter as cis-regulatory elements.

8. The gene transfer system of claim 3, wherein two or three of the components (a) the ligand oligopeptide, (b) the polycationic polypeptide, and (c) the endosomal nucleosome releasing oligopeptide are present as a fusion protein.

9. The gene transfer system of claim 2, wherein the endocytosome release oligopeptide element is selected from the group consisting of: HA20 or VP-1.

10. An oligopeptide which binds to Tie2 receptor and comprises the amino acid sequence: SEQ ID NO: 1.2 or 3.

Images