Inhibition lung cancer transfer and lung transfer tumor gene medicament

A transgene and drug technology, applied in the field of molecular medicine and disease prevention and molecular pharmacology, can solve the problems of low concentration of anti-tumor angiogenesis factors and inability to better play the purpose of treating tumors in target organs

Inactive Publication Date: 2011-10-05
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

[0075] Preparation of rAAV2 / 2-eGFP

[0076] The composition of the expression frame 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 ITRs.

[0077] Prepare recombinant virus by calcium phosphate-free helper virus multi-plasmid method: co-transfect 293 cells with eGFP expression plasmid, AV helper plasmid, and rAAV2 helper plasmid, harvest cells after 60-72 hours, and use sodium deoxycholate (0.5%) Cleave with benzonase (30u / ml), centrifuge (3000g 30min) to remove cell debris, and cesium chloride gradient centrifuge to obtain recombinant virus rAAV2 / 2-eGFP, which is purified by chromatography.

Embodiment 2

[0079] 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

[0080] Referring to the steps in Example 1, the rAAV2 helper plasmids were replaced with rAAV1, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, and rAAV11, and the remaining operations were the same as in Example 1 to obtain 11 different serotypes of rAAV viruses.

Embodiment 3

[0082] Screening for lung-tropic rAAV serotypes

[0083] The titers of the various serotypes of rAAV obtained in Example 2 were determined by Real-PCR method. The primers used were designed according to the sequence of WPRE, respectively WPRE-F (5'-TGGCGTGGTGTGCACTGT) and WPRE-R (5'-GTTCCGCCGTGGCAATAG).

[0084] LLC cells or HLEC cells were seeded into 24-well plates and cultured overnight. Add the various serotype rAAV viruses that contain embodiment 2 and obtain respectively (MOI 10 4 ) serum-free medium, after incubation for 5 hours, it was replaced with normal medium. After 72 hours, the cells were observed with a fluorescent microscope, and the cells were sorted with a flow cytometer, and the transduction efficiency was calculated (n=3).

[0085] Table 3 Transduction efficiency of different serotypes of rAAV to LLC cells or HLEC cells

[0086] rAAV serotype

[0087] rAAV2 / 4

[0088] It can be seen that the transduction efficiency of various serotyp...

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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, in particular to a gene drug based on recombinant adeno-associated virus (rAAV) and tumor angiogenesis inhibitory gene, which can target lung tissue and effectively inhibit the metastasis of lung cancer and lung metastases. Background technique [0002] Primary bronchial lung cancer (abbreviated as lung cancer) is the most common primary malignant tumor of the lung. It ranks first in the statistics of the incidence of tumors and the mortality of patients due to tumors in the world, accounting for about 18% of the total number of cancer patients. Currently, there are many clinically used drugs for the treatment of lung cancer, including paclitaxel (paclitaxel), a topoisomerase II inhibitor, etoposide (etoposide), a platinum compound, carboplatin, and docetaxel. (docetaxel) etc. are used in the treatment of non-small cell lung ...

Claims

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Application Information

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