Construction method and applications of targeting oncolytic-adenovirus carier Ad-TD-gene

A virus vector and construction method technology, applied in the fields of biotechnology and gene therapy, can solve the problems of increasing the loading of foreign genes and affecting the anti-tumor effect of oncolytic adenovirus, so as to achieve the effect of good curative effect and safety, and increase the specificity

Active Publication Date: 2010-02-10
BEIJING BIO TARGETING THERAPEUTICS TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It was previously believed that the gene in the E3 region of adenovirus has no effect on the replication of adenovirus in vitro, and deleting this region can increase the ability to load foreign genes, but recently we found that the E3B region of adenovirus seriously affects the anti-tumor effect of oncolytic adenovirus, deleting The gene in this region obviously accelerates the body's clearance of adenovirus, but the deletion of E3gp-19K in the E3 region leads to enhanced anti-tumor effect of the virus

Method used

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  • Construction method and applications of targeting oncolytic-adenovirus carier Ad-TD-gene
  • Construction method and applications of targeting oncolytic-adenovirus carier Ad-TD-gene
  • Construction method and applications of targeting oncolytic-adenovirus carier Ad-TD-gene

Examples

Experimental program
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Embodiment 1

[0036] Example 1. Construction of tumor-targeting adenoviral vectors Ad-TD-gene, Ad-TD-hCCL21 and their control viral vector Ad-TD-RFP.

[0037] 1. Construction of tumor targeting adenoviral vector Ad-TD-gene

[0038] (1) First use the PCR method to obtain the gene fragments on both sides of the sequence E1A-CR2 to be transformed. The upstream sequence is called the left arm, and the downstream sequence is called the right arm. Use genetic engineering methods to place the left arm and the right arm in the same order as the viral genes Connect to the plasmid vector pSuperShuttle to build the E1A-CR2 shuttle vector;

[0039] (2) Transform the Ad5 adenovirus vector and the shuttle vector into BJ5183 bacteria at a ratio of 1:2 to 10 for homologous recombination; use PCR to identify positive recombinant bacteria, extract the recombinant plasmid, and transfect it into 293 cells to obtain E1A-CR2 deleted Ad5R-CR2 viral vector;

[0040] (3) Construct the E1B19K shuttle vector with t...

Embodiment 2

[0048] Example 2. The endogenous promoter of the tumor-targeting adenoviral vector Ad-TD-gene expresses the specific expression of the reporter gene RFP in the cell

[0049] 293 cells were inoculated into a six-well plate, and when the cells grew to 75%, Ad-TD-RFP was injected at 1×10 5 The dose of pt / cell was used to infect 293 cells. After culturing for 48 hours, the expression of red fluorescent protein was observed under a fluorescent microscope. The results are shown in the attached figure 2 shown.

Embodiment 3

[0050] Example 3, Detection of the Killing Ability of Tumor Targeting Adenovirus Vectors Ad-TD-gene and Ad-TD-hCCL21 to Common Human Tumor Cells

[0051] HCT116 (colorectal cancer) cells, PT45 (pancreatic cancer) cells, H460 (lung cancer) cells, A549 (lung cancer) cells, SUIT2 (pancreatic cancer) cells in the logarithmic growth phase were collected, counted, and seeded at 2000 cells / well in in a 96-well plate. After 12 hours, the cells were attached and the viruses dl1520, Ad-TD, Ad-TD-hCCL21 and Ad-TD-mCCL21 (mCCL21, mouse CCL21 gene) were diluted. According to the highest concentration 1×10 4 pt / cell was added to a 96-well plate, and each column was decreased by 10 times, and the 10th column was a blank control. Six days later, MTS method detection (Buttke TM, J Immunol Methods, 1993, 157 (1-2): 233-40), converted into EC50 (that is, the amount of virus required for each virus to kill 50% of tumor cells, the lower the EC50 , indicating that the virus killing ability is st...

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Abstract

The invention relates to a construction method and applications of targeting oncolytic-adenovirus carier Ad-TD-gene. The carrier of the oncolytic-adenouirus is Ad5, the adenovirus-5 in Subcategory C;three internal genes of the adenovirus, including E1A-CR2, E1B19K and E3gp-19K, are deleted; an E3B gene is retained, and an E3gp-19K promoter expression exogenous therapeutic gene is retained; and any antigen gene which is helpful to treating tumors or used for infectious disease vaccines can be inserted into the carrier. The Ad-TD-gene has the advantages of specificity, high safety and high efficiency, can carry various antigen genes treating tumors, and can be used for treating various solid tumors. By utilizing the human chemotactic factor CCL 21 gene as the therapeutic gene, the Ad-TD-hccL21 has the effects of tumor targeting and tumor resistance, lays foundation for introducing targeting genetic engineering medicines towards clinical applications, and provides an effective therapy for patients with tumors.

Description

1. Technical field: [0001] The invention relates to the fields of biotechnology and gene therapy, in particular to a construction method and application of a tumor-targeting adenoviral vector Ad-TD-gene. 2. Background technology: [0002] Malignant tumors are serious diseases that threaten human health, especially advanced malignant tumors. Chemotherapy and radiotherapy are commonly used methods in the clinical treatment of advanced tumors. Although standard chemotherapy and radiotherapy have been used clinically for decades, the overall survival rate of most clinically advanced patients has not been significantly improved. Therefore, in theory and clinical practice, not only innovative tumor treatment methods are needed, but also have better selectivity and clearer mechanism of action on tumor cells, and at the same time will not produce antagonism with the commonly used therapeutic drugs. [0003] Viruses can infect human cells through various mechanisms, replicate in ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K48/00A61K39/00A61K39/02A61K39/106A61K39/21A61K39/29A61P35/00A61P31/18A61P31/04A61P33/12A61P31/12
Inventor 王尧河姜国忠王鹏举尼克·莱蒙
Owner BEIJING BIO TARGETING THERAPEUTICS TECH
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