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Tie2 receptor mediated gene transfer system for targeted tumor gene therapy

A gene transfer and targeting technology, applied in the fields of molecular biology and gene therapy, can solve the problems of high price, unrealistic large-scale preparation, and inability to meet clinical applications

Inactive Publication Date: 2007-07-04
NEWORGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although natural ligands can effectively bind Tie2 to achieve a good targeting effect, the purification of natural Tie2 ligands is not only technically complicated, but also large-scale preparation is unrealistic, so it cannot meet clinical applications.
Only a small number of human Tie2 ligands are currently available for scientific experiments, and they are expensive
In addition, the artificially prepared anti-Tie2 antibody is a non-humanized macromolecular protein, which is a foreign protein antigen molecule for the human body, has strong immunogenicity, and is difficult to be used in clinical practice.

Method used

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

Examples

Experimental program
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Effect test

preparation example Construction

[0071] Preparation method of targeted non-viral vector

[0072] Usually, the above-mentioned (a), (b), (c) components or their complexes are mixed together to obtain the targeting non-viral vector of the present invention. Wherein, the mixing ratio of (a), (b) and (c) components is usually 0.5-1:1-2:0-1, preferably 0.8-1:1-1.5:0.5-1.

[0073] In addition, when administered in the form of (a)-(b) complex and (b)-(c) complex, the mixing ratio of (a)-(b) complex: (b)-(c) complex Usually 0.8-1.2:0.8-1.2, preferably 0.9-1.1:0.9:1.1.

[0074] exogenous DNA

[0075] The exogenous DNA that can be used in the present invention is not particularly limited, and can be various therapeutic or preventive DNAs, such as target genes, antisense oncogenes, anticancer genes, suicide genes, apoptosis genes, cytokine genes, or A combination thereof, or a eukaryotic expression vector DNA containing the above genes. Proto-oncogene antisense sequences include proto-oncogene (ras H 、ras K 、ras ...

Embodiment 1

[0090] Example 1 Chemically synthesized polypeptide and its mass spectrometry analysis

[0091] In this example, GA3, GA4 and GA5 were prepared by artificial synthesis.

[0092] Taking the polypeptide GA3 [SEQ ID NO: 1] as an example, its synthesis is carried out on a polypeptide synthesizer by a solid-phase synthesis method. The used carrier resin is HMP amino resin, the amount of amino acid is 1 mmol, ie amino acid:resin=4:1. According to the amino acid sequence of the polypeptide GA3 [SEQ ID NO: 1], each amino acid component is sequentially added for solid-phase synthesis. After the peptide is synthesized, cut it according to the steps recommended by PE company, filter the reacted mixture with a G4 glass sand funnel to remove the resin, evaporate the filtrate to 1ml under low pressure at room temperature, add 50ml pre-cooled ether to precipitate the peptide, and overnight at 4°C , filtered through a G6 glass sand funnel, and vacuum freeze-dried to obtain the crude polypep...

Embodiment 2

[0095] Example 2 Specific Binding Test of GA3 and Tie2 Receptor on the Surface of SMMC7721 Cells Stably Expressing Tie2

[0096] (1) Establishment of cell lines stably expressing the extracellular region of Tie2

[0097] (1) The human cell line SMMC7721 negatively expressing Tie2 was screened out by conventional Western blot method as follows:

[0098] Collect one plate (9 cm) of different well-growing tumor cell lines, wash three times with PBS, add 1ml PBS, scrape off the cells with a spatula, collect them in a 1.5ml centrifuge tube, 8000rpm, 1min, remove the supernatant, add protein to lyse 100 μl of solution (containing 1 μg of protease inhibitor), lysed on ice for 30 minutes, centrifuged at 10,000 rpm for 10 minutes, transferred the supernatant to a new tube, and obtained the total cell protein. Take 20 μl of protein solution and add 4 μl of sample buffer, cook for 10 minutes, centrifuge at 10,000 rpm for 10 minutes, take supernatant for protein electrophoresis, transfer...

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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

technical field [0001] The invention relates to the fields of molecular biology and gene therapy. Specifically, it relates to a gene transfer system based on a ligand oligopeptide binding to the angiopoietin receptor Tie2. Through receptor-mediated endocytosis, the transfer system can target exogenous DNA into tumor neovascular endothelial cells and tumor cells expressing Tie2 receptors, so as to achieve the purpose of treating tumors. Background technique [0002] PCT application PCT / CN97 / 00106 (WO98 / 18951) discloses a novel receptor-mediated gene transfer system for targeted tumor gene therapy, which discloses three kinds of receptors targeting EGFR, IGF I / II R and VEGFR Body-mediated gene transfer system for tumor therapy. However, because the receptors on the surface of different tumor cells are diverse, with varying amounts of expression, or even no expression of some receptors at all, only three systems cannot solve all problems. [0003] In 1992, Partanen J et al. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P35/00C07K7/00
Inventor 顾健人吴向华徐宇虹李宗海
Owner NEWORGEN
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