Tumourolytic anticancer recombined adenovirus with tumour target direction and idioctonia

A technology of recombinant adenovirus and adenovirus, applied in antitumor drugs, virus/phage, drug combination, etc., can solve problems such as low infection rate, achieve enhanced infection, dose-expanding effect and anti-cancer ability, inhibit growth and spread Effect

Inactive Publication Date: 2006-06-28
陈智博
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Adenovirus mainly relies on the adenovirus receptor (CAR) on the cell surface to infect cells, an

Method used

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  • Tumourolytic anticancer recombined adenovirus with tumour target direction and idioctonia
  • Tumourolytic anticancer recombined adenovirus with tumour target direction and idioctonia
  • Tumourolytic anticancer recombined adenovirus with tumour target direction and idioctonia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Recombinant construction of oncolytic adenovirus.

[0058] There are ten different oncolytic recombinant adenoviruses in the present invention. The specific production methods and steps of various viruses have been described in the previous technical solutions and will not be repeated here. Figure 1 is a schematic diagram of the genome DNA of these ten recombinant adenoviruses, mainly indicating the sites and genes of the genetic modification of ad5 in the present invention.

Embodiment 2

[0059] Example 2: Detection of self-replication ability of recombinant adenovirus by plaque method.

[0060] In order to evaluate the replication ability of the recombinant adenovirus of the present invention, we used wild-type ad5d1309 (its E3 has partial deletion and insertion mutation) as a control, and infected human liver cancer cell lines Hep3B, Hep3B, Cervical cancer cell line Hela, lung cancer cell line H661, normal liver cell Chang liver, normal breast epithelial cell HMEC and normal lung fibroblast WI38. After three days, cells were harvested. After three freezing and thawing at -80°C and 30°C to release the virions, the cell supernatant was used to infect HEK 293 cells to determine the viral load (pfu) by phagocytosis. In the same cell line, the comparison of the amount of virus produced by the recombinant virus and the wild-type virus can indicate the replication ability of the recombinant virus. Figure 2 lists the replication capabilities of representative recom...

Embodiment 3

[0061] Example 3: Detecting the effect of adding RGD short peptide to the fibrin terminal section on the infectivity of virus tumor cells.

[0062] We selected human bladder cancer cell line T24, ovarian cancer cell line HEY and pancreatic cancer cell line Hs7667 to compare the infectivity of adE1Adl2p and adE1Adl2p.RGD. All three types of cancer cells have a moderate amount of adenovirus receptor CAR and a large amount of Cancer cells were infected with the same dose of the two viruses (MOI: 50). After 5 hours, the cells were harvested, and viral DNA was extracted from the cells. Then the 1Kb MLP fragment in the adenovirus DNA was amplified by quantitative PCR. The amount of PCR DNA obtained is shown in Figure 3. In these cancer cells, the infectivity of the recombinant adenovirus adE1Adl2p.RGD with RGD short peptide at the end of fibrin was 4-6 times that of the control virus adE1Adl2p. Obviously, the RGD short peptide can effectively enhance the recombinant adenovirus ...

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Abstract

The invention offers two sets of tumor-dissolving recombination adenovirus. One set is produced by rite-directed mutagenesis in virus E1A gene. The other set produced by replacing E1A promoter with tumor specificity promoter. The fiber end of parts of the recombination adenovirus is added RGD short peptide. And parts of are carried HSV1-tkm. It is proved by experiment that the two sets of virus can strongly infect tumor cell, do a great deal replication and breeding, and finally dissolve cell. The released filial generation adenovirus can infect around tumor cell again. Thus dosage effect and against cancer capability of virus gene can be enlarged by breeding infecting cycle. The tow sets of tumor-dissolving recombination adenovirus appearances strong against cancer effect in animal subcutaneous, human liver and lung tumor model test.

Description

technical field [0001] The invention belongs to the field of anti-cancer, and relates to malignant tumor gene therapy and oncolytic virus therapy. The recombinant adenovirus has strong oncolytic ability by genetically modifying the E1A gene of the adenovirus. At the same time, genetically modifying the fiber gene (fiber) of the adenovirus leads to tumor targeting of the oncolytic recombinant adenovirus. When carrying tumor suicide genes, these recombinant adenoviruses showed stronger anticancer effects. These oncolytic, tumor-targeting, and tumor-suicidal recombinant adenoviruses showed strong anti-cancer capabilities in experiments on various human tumor models established in mice. In normal cells, these recombinant adenoviruses have no self-replicating ability, and their pathogenicity to normal tissues is very slight, which ensures the safety of clinical application. Background technique [0002] Cancer has become the number one fatal disease in modern society, especial...

Claims

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Application Information

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IPC IPC(8): C12N7/01A61K48/00A61P35/00C12N15/861
Inventor 陈智博
Owner 陈智博
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