Method for selectively replicating replication-defective adenovirus and application

A replication-defective, adenovirus technology, applied in the field of biomedicine, can solve the problems of danger, ineffective gene therapy, low activity, etc., and achieves the effect of overcoming safety problems and broad application prospects

Inactive Publication Date: 2010-11-10
SHANGHAI FIRST PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The effectiveness and safety of adenovirus have always been the bottleneck restricting the wide application of adenovirus in clinical practice of gene therapy
However, there are still some problems in the application of adenoviral vectors, especially in vivo applications: ①Adenoviral vectors mediate the expression of foreign genes for a short time
②Exogenous gene expression level is still not ideal
Although adenoviral vectors can mediate short-term high-level expression of foreign genes, the current therapeutic genes are not effective enough, and most of the adenoviral vectors used for gene therapy are replication-deficient vectors, which cannot replicate in vivo, and the dosage is too high. The general assembly will lead to acute toxicity, so the expression level of exogenous genes is still not ideal for tumor gene therapy, and there is almost no room for increasing the expression level of exogenous genes by increasing the dosage
③Toxic and side effects caused by non-specific distribution and virus leakage
However, there are still the following defects: ① most tumor tissue-specific promoters have low activity, and the effectiveness of gene therapy will decrease while the safety is improved; ② the stringency of tumor tissue-specific promoters is relative, if Leakage of a large amount of adenovirus into circulating blood still poses potential danger; ③The construction process is cumbersome and requires one virus one construction

Method used

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  • Method for selectively replicating replication-defective adenovirus and application
  • Method for selectively replicating replication-defective adenovirus and application
  • Method for selectively replicating replication-defective adenovirus and application

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Isolation of tumor-specific promoters by PCR reaction and cloning

[0062] A. The regulatory sequence at the 5' end of telomerase

[0063]During natural DNA replication, the ends of chromosomes shorten with each cell division. The integrity of telomeres at the end of chromosomes is crucial to the stability of chromosomes. The integrity of telomeres is maintained by telomerase. Telomerase is inactive in more than 99% of normal cells and is reactivated in more than 90% of tumor cells. Experiments have shown that the activity of telomerase is controlled by its promoter. The promoter of telomerase (TERT) is active in most tumors but not in normal cells. Thus, the promoter of TERT becomes a tumor-selectively activated promoter. In order to isolate the telomerase promoter, the present invention uses PCR technology to amplify the promoter (sequence one) using human cell DNA as a template, and insert it into the plasmid pEGFP-1 (purchased from Clontech Company) to drive th...

Embodiment 2

[0079] A eukaryotic expression vector carrying a tumor-specific promoter regulating the E1A gene, an essential element for adenovirus replication, was constructed.

[0080] The tumor-specific promoter (TSP) isolated in Example 1 was embedded into a eukaryotic expression vector by genetic engineering, and used to control the expression of the early gene E1A necessary for adenovirus replication. The characteristic of the plasmid is that the adenovirus replication essential element E1A gene carried by it is regulated by a tumor tissue-specific promoter. The structure of the eukaryotic expression vector used in the present invention is schematically shown as: Plasmid-TSP-E1A, wherein Plasmid is any eukaryotic expression plasmid (such as: pcDNA3.1); TSP is the tumor-specific isolated in Example 1 promoter and other tumor-specific promoters not mentioned.

Embodiment 3

[0082] To construct an adeno-associated virus (rAAV) vector carrying a tumor-specific promoter regulating the essential element E1A gene for adenovirus replication.

[0083] The tumor-specific promoter (TSP) isolated in Example 1 was embedded into the shuttle plasmid of the adeno-associated virus by genetic engineering, and used to control the expression of the early gene E1A necessary for the replication of the adenovirus. The characteristic of the plasmid and virus is that the adenovirus replication essential element E1A gene carried by them is regulated by a tumor tissue-specific promoter. The shuttle plasmid structure of the adeno-associated virus used in the present invention is schematically shown as pSNAV-TSP-E1A, wherein, pSNAV is a general-purpose shuttle plasmid for packaging different serotypes of AAV, and can be used for packaging any serotype of adeno-associated virus; TSP is Example 1 Tumor-specific promoters isolated in and other tumor-specific promoters not men...

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Abstract

The invention belongs to the field of bio-medicament, and relates to a method for selectively replicating replication-defective adenovirus and application. Necessary element E1A genes for replicating the replication-defective adenovirus are controlled by tumor cells or tumor tissue-specific promoters, carried by eukaryotic expression plasmids or recombinant gland related virus vectors and provided reversely in tumor local. The adenovirus can effectively kill the tumor cells by synergy of tumor dissolution action, carried treatment gene action and E1A cancer inhibiting action and basically has no harm to normal tissues or cells. The method can conveniently and quickly endow any replication-defective adenovirus for tumor gene therapy with selective proliferation and replication capabilities in the tumor local. The method can be separately used or combined with the conventional tumor therapy method, remarkably improve the tumor therapy effect and reduce the side effect.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a replication-deficient adenovirus, in particular to a method for selectively replicating a replication-defective adenovirus, and in particular to a method for in situ selection of a replication-defective adenovirus for tumor gene therapy Methods and applications of sexual reproduction. Background technique [0002] Malignant tumors are the most important diseases that endanger human health. Traditional methods such as surgery, chemotherapy, and radiotherapy are still unable to significantly improve the relative helplessness of people with this type of disease. After 20 years of development, gene therapy has attracted more and more attention as a new means of treating tumors. As of January 2009, among the 1472 gene therapy clinical trials that have been implemented around the world, the gene therapy used for tumor treatment There are 960 items, accounting for 65.2%. Adenovirus (Adenovi...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/79C12N15/86A61K48/00A61P35/00
Inventor 王慧萍韦芳
Owner SHANGHAI FIRST PEOPLES HOSPITAL
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