Mosaic tumor specific gene expression regulation kit and application thereof

A tumor-specific, gene expression technology, applied in the field of new tumor gene therapy targeted expression technology and gene therapy system, can solve the problems of mRNA stability and expression efficiency that need to be improved

Inactive Publication Date: 2011-05-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the current targeted expression technology of tumor therapeutic genes, more focus is on the transcriptional targeted expression level, and the mRNA stability and expression efficiency under its regulation need to be improved.

Method used

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  • Mosaic tumor specific gene expression regulation kit and application thereof

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Experimental program
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Effect test

Embodiment 1

[0049] Isolation and Cloning of Tumor-Specific Gene Expression Regulatory Cassette Regulatory Elements

[0050] A. hTERT gene promoter

[0051] Telomerase is an RNA-dependent DNA polymerase. The activation of telomerase is an important mechanism for cells to obtain immortalization and malignant transformation. Studies have shown that telomerase is positively expressed in more than 85% of tumor cells, but negative in almost all adult human cells. The telomerase catalytic subunit encoded by human telomerase reverse transcriptase gene hTERT is the decisive factor of telomerase activity. The activity of hTERT itself is mainly regulated at the transcriptional level, and the transcriptional activity of the hTERT gene promoter plays an important role in this process. Since the hTERT promoter is only transcriptionally active in tumor cells, hTERT is an ideal transcriptional regulatory element for targeted expression of therapeutic genes in tumor cells. The embodiment of the present...

Embodiment 2

[0061] Construction of chimeric tumor-specific gene expression regulatory cassettes

[0062] The various tumor-specific gene expression regulatory cassettes isolated and cloned above need to be arranged and combined in a certain order to construct a chimeric tumor-specific gene expression regulatory cassette. The embodiment of the present invention uses the basic principle of molecular cloning technology to design and chemically synthesize multiple cloning sites MCS (Multiple Cloning Sites, MCS) (SEQ No. 6), after double digestion with PacI and XhoI, insert into the same double digestion The pORF-MCS (Invivogen) constitutes the backbone plasmid pBA-MCS ( figure 1 A). The hTERT gene promoter sequence confirmed by sequencing in Example 1 was double-digested with EeoRI and HindIII, bFGF-25'UTR was double-digested with HindIII and BamHI, WRE was double-digested with HindIII and SalI and blunt-ended, EGFR The 3'UTR was digested with BglII, BGHpolyA was digested with BglII and Xho...

Embodiment 3

[0064] Establishment of Chimeric Tumor-Specific Gene Expression Regulatory Cassette-Regulated Reporter Genes

[0065] Whether the chimeric tumor-specific gene expression regulatory cassettes contained in the expression plasmids phTUW-MCS and phTUWR-MCS constructed above can play a role needs to be further analyzed and verified by reporter genes in cell lines. In the embodiment of the present invention, the enhanced green fluorescent protein gene EGFP was excised from the plasmid pFUGW (Addgene) by NcoI and XbaI double enzyme digestion, and inserted into the expression plasmids phTUW-MCS and phTUWR-MCS after the same digestion, respectively, to construct a qualitative gene reporter Plasmids phTUW-EGFP and phTUWR-EGFP ( figure 1 C). The above-mentioned reporter gene was used to further transfect various tumor cell lines to confirm its tumor-specific dual regulatory gene expression effect. Compared with normal cells (such as normal liver cell L-02), the reporter gene EGFP regul...

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Abstract

The invention belongs to the technical field of biomedicines, and in particular provides a mosaic tumor specific gene expression regulation kit, which comprises a tumor specific gene promoter, a 5' untranslated region of a tumor specific translational control component, a polyclonal site, a gene expression enhancer and a polyadenylic acid signal region, wherein the tumor specific gene promoter, the 5' untranslated region of the tumor specific translational control component, the gene expression enhancer and the polyadenylic acid signal region are connected in turn; and the polyclonal site is positioned between the 5' untranslated region of the tumor specific translational control component and the polyadenylic acid signal region and is independent of the gene expression enhancer. The mosaic tumor specific gene expression regulation kit effectively regulates the expression of tumor therapy genes to limit the genes in tumor cells, and obviously improves the specificity of the expression of the therapy genes so as to effectively improve the safety of tumor gene therapy and reduce the harm of the tumor gene therapy to normal tissues and cells.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and specifically relates to a novel targeted expression technology for tumor gene therapy, that is, by constructing a chimeric tumor-specific gene expression control box, using the control box to regulate the expression of tumor treatment genes, and Therefore, a tumor-specific gene therapy system is constructed. Background technique [0002] With the development of modern genetics and genomics, gene therapy as a new disease treatment has become possible. Gene therapy is to directly intervene in the occurrence, development and process of diseases through manipulation and intervention at the gene level. Compared with other existing treatment methods and strategies, the advantage of gene therapy is that it can directly use people's new discoveries and new understandings of the etiology and pathogenesis of diseases at the molecular level, as well as new technologies in the field of molecular bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/63C12N7/00C12N7/01A61K48/00A61P35/00
Inventor 田聆方煜翔陈金中薛京伦
Owner FUDAN UNIV
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