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Inhibition lung cancer transfer and lung transfer tumor gene medicament

A drug and gene technology, applied in the field of gene medicine based on recombinant adeno-associated virus and tumor angiogenesis inhibitory gene, can solve the problems of not being able to better play the purpose of target organ tumor treatment, low concentration of anti-tumor angiogenesis factors, etc.

Inactive Publication Date: 2008-06-18
刁勇 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In addition, the studies on anti-tumor angiogenesis factor gene therapy all adopt the mode of systemic administration such as intramuscular injection and intravenous injection, and the concentration of anti-tumor angiogenesis growth factor generated in the target organ is low, which cannot better play the therapeutic purpose of target organ tumor

Method used

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  • Inhibition lung cancer transfer and lung transfer tumor gene medicament
  • Inhibition lung cancer transfer and lung transfer tumor gene medicament
  • Inhibition lung cancer transfer and lung transfer tumor gene medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Preparation of rAAV2 / 2-eGFP

[0075] The composition of the expression cassette of the eGFP expression plasmid is as follows: the promoter is CMV enhancer / chicken β-actin promoter, eGFPcDNA, polyA, WPRE. Both ends of the expression box are AAV2 ITR.

[0076] Recombinant virus was prepared by calcium phosphate helper-free virus multi-plasmid method: eGFP expression plasmid, AV helper plasmid, and rAAV2 helper plasmid were co-transfected into 293 cells, 60-72 hours later, the cells were harvested, and sodium deoxycholate (0.5%) It was lysed with benzonase (30u / ml), centrifuged (3000g for 30min) to remove cell debris, and cesium chloride gradient centrifugation to obtain recombinant virus rAAV2 / 2-eGFP, which was purified by chromatography.

Embodiment 2

[0078] rAAV2 / 1-eGFP, rAAV2 / 3-eGFP, rAAV2 / 4-eGFP, rAAV2 / 5-eGFP, rAAV2 / 6-eGFP, rAAV2 / 7-eGFP, rAAV2 / 8-eGFP, rAAV2 / 9-eGFP, rAAV2 / Preparation of 10-eGFP and rAAV2 / 11-eGFP

[0079] Referring to the steps in Example 1, the rAAV2 helper plasmids were replaced with rAAV1, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, and the remaining operations were the same as in Example 1, to obtain 11 different serotype rAAV viruses.

Embodiment 3

[0081] Screening of lung tropism rAAV serotype

[0082] The Real-PCR method was used to determine the titers of various serotypes of rAAV obtained in Example 2. The primers used are designed according to the WPRE sequence and are WPRE-F (5'-TGGCGTGGTGTGCACTGT) and WPRE-R (5'-GTTCCGCCGTGGCAATAG).

[0083] Inoculate LLC cells or HLEC cells into a 24-well plate and culture overnight. Each serotype rAAV virus (MOI 10 4 ) Serum-free medium, after 5 hours of incubation, replace with normal medium. After 72 hours, observe with a fluorescence microscope and perform cell classification with a flow cytometer, and calculate the transduction efficiency (n=3).

[0084] Table 3 Transduction efficiency of different serotypes of rAAV on LLC cells or HLEC cells

[0085] rAAV serotype

[0086] rAAV2 / 4

[0087] It can be seen that the transduction efficiency of various serotypes of rAAV to LLC cells or HLEC cells are different, but in comparison, rAAV2 / 1, rAAV2 / 2 and rAAV2 / 5 have...

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Abstract

The invention relates to materia medica, molecular medicine and disease prevention fields, in particular to a gene medicine based on recombinant adeno-associated virus and tumor angiogenesis inhibiting gene. The medicine can target at lung tissue and inhibit effectively cancer metastasis and the occurrence of pulmonary metastases.

Description

Technical field [0001] The present invention relates to the fields of molecular pharmacology, molecular medicine and disease prevention and treatment, and in particular to gene drugs based on recombinant adeno-associated virus (rAAV) and tumor angiogenesis inhibitor genes, which can target lung tissue and effectively inhibit lung cancer metastasis And the occurrence of lung metastases. Background technique [0002] Primary bronchial lung cancer (referred to as lung cancer) is the most common primary malignant tumor of the lung. It ranks first in the world's tumor incidence and tumor mortality statistics, accounting for about 18% of the total number of cancer patients. At present, there are many kinds of lung cancer treatment drugs commonly used clinically, including paclitaxel (paclitaxel), topoisomerase II inhibitor etoposide, platinum compound carboplatin and docetaxel (docetaxel) and others are used to treat non-small cell lung cancer (NSCLC). Although they have certain curati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P35/04
Inventor 刁勇许瑞安蔡克霞
Owner 刁勇
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