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Construction and application of tumour targeting gonad correlation viral vectors

A technology of tumor targeting and viral vectors, which can be used in anti-tumor drugs, introduction of foreign genetic material using vectors, gene therapy, etc., and can solve the problems of safety, low targeting and transfection efficiency, and lack of host-range cell specificity to achieve high targeting, improve targeting, and inhibit tumor growth

Inactive Publication Date: 2008-05-07
ZHEJIANG SCI-TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the wide host range and lack of tissue or cell specificity of AAV2, there are still disadvantages of safety, targeting and low transfection efficiency when it is used as a vector for preclinical and clinical treatment trials.

Method used

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  • Construction and application of tumour targeting gonad correlation viral vectors
  • Construction and application of tumour targeting gonad correlation viral vectors
  • Construction and application of tumour targeting gonad correlation viral vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Construction of tumor-targeting adeno-associated virus rAAV-hTERT-hTRAIL and control virus rAAV-hTERT-EGFP:

[0038] 1. Design primers:

[0039] hTERT:

[0040] sense: 5'-TGGCCCCTCCCTCGGGTTAC-3'

[0041] antisense (EcoR I): 5'-CCC GAATTC CGCGGGGGTGGCCGGGGCCA-3'

[0042] hTRAIL:

[0043] sense (EcoR I): 5'-CTC GAATTC ATGGCTATGATGGAGGTCCAG-3'

[0044] antisense (Hind III): 5'-CCT AAGCTT CAGGTC AGTTAGCCAACTAA-3'

[0045] EGFP:

[0046] sense (EcoR I): 5'-CTC GAATTC ATGGTGAGCAAGGGCGAGGAG-3'

[0047] antisense (Hind III): 5'-CCT AAGCTT TTATCTAGATCCGGTGGATC-3'

[0048] 2. Construction of related plasmids:

[0049] 1. Construction of pAAV-hTERT-hTRAIL:

[0050] 1), using the specific primers at both ends of the above hTERT promoter: 5'-GCTCCCAGTGGATTCGCG-3'(sense) and 5'-CCC GAATTC CGCGGGGGTGGCCGGGGCCA-3' (antisense, containing EcoR I restriction site), using the total DNA genome extracted from human liver cancer cell line SMMC-7721 as a template,...

Embodiment 2

[0060] Example 2. Analysis of the specific expression of the reporter gene EGFP mediated by the hTERT promoter in tumor cells and normal cells carried by an adeno-associated virus vector:

[0061] Cells were inoculated into 6-well plates, and when the cells grew to about 70%, rAAV-hTERT-EGFP was mixed with 5×10 4 / cell infection normal cell L02 and three kinds of tumor cells SMMC-7721, SGC7901, A549, after cultivating for 2 days, observe the expression of green fluorescence under the fluorescence microscope (see Figure 3, Figure 3 is the contrast virus rAAV-hTERT-EGFP transfection tumor Comparison of EGFP expression in cells and normal cells). The results showed that rAAV-hTERT-EGFP infection could target expression of EGFP in tumor cells, but there was little expression of green fluorescence in normal cells; that is, the transcriptional activity of hTERT promoter was very low in normal cells, but in tumor cells Mediate specific high expression of EGFP gene.

Embodiment 3

[0062]Example 3. Detection of tumor cell killing ability of tumor-targeted adeno-associated virus rAAV-hTERT-hTRAIL:

[0063] The survival rate of cells after virus treatment was detected by MTT method (Cancer Research, 1989, 49(17): 4785-90). The steps are as follows: the normal cell L02 and three kinds of tumor cells SMMC-7721, SGC7901, A549 were plated in 96-well plates at the amount of 5000 cells per well, and cultured for 24 hours, respectively, in 5×10 4 The cells were infected with two viruses (rAAV-hTERT-hTRAIL and rAAV-hTERT-EGFP) per cell, and the cell viability was measured by MTT after 1, 2, 3, 4, and 5 days respectively. The details are as follows: Remove the virus-containing culture solution and replace it with a normal culture solution containing 5 mg / ml MTT. After cultivating for 4 hours, remove the MTT-containing culture solution, crack with DMSO, shake well, and then measure the absorbance at 655 nm as Absorbance at 595 nm was measured for reference.

[006...

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Abstract

The invention discloses a construction method of a tumor targeting adeno-associated virus vector system rAAV-hTERT-gene: a tumor specific hTERT is used to start the foreign genes carried by a sub-control-gland-associated virus to express to construct an adeno-associated virus vector system rAAV-hTERT-gene which has tumor targeting and can be inserted into a therapeutic gene with anticancer effect at will. The invention also discloses the tumor targeting adeno-associated virus rAAV-hTERT-hTRAIL constructed by the above method: the virus is an anti-tumor active hTRAIL gene with wide selectivity carried by a tumor targeting adeno-associated virus vector system rAAV-hTERT-gene. The invention has the advantages of constructing safe, high efficient tumor targeting adeno-associated viruses.

Description

technical field [0001] The present invention belongs to the fields of biotechnology and gene therapy, and in particular relates to a targeted adeno-associated virus vector rAAV-hTERT-hTRAIL specifically expressed in tumor cells and its construction method and application. Background technique [0002] For most types of cancer, traditional therapies such as surgery, radiotherapy, chemotherapy, etc. are still used at present, but in the face of many advanced malignant tumors, traditional therapies are helpless, mainly manifested in low cure rate, high recurrence rate and high mortality , The prognosis is poor. Gene therapy is a biological high-tech solution to treat diseases by modifying genes. People are very enthusiastic about gene therapy for tumors, and 66% of gene therapy clinical trials are used for tumor treatment. So far, tumor gene therapy research has made important progress; for malignant tumors, gene therapy can take advantage of the molecular level differences be...

Claims

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Application Information

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IPC IPC(8): C12N15/86A61K48/00A61P35/00
Inventor 王毅刚黄芳张义玲姜永厚钱程刘新垣
Owner ZHEJIANG SCI-TECH UNIV
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