Gene engineering adenovirus and it application

A technology for recombinant adenoviruses and viruses, which is applied in the directions of viruses/phages, medical preparations containing active ingredients, biochemical equipment and methods, etc., and can solve problems such as high target specificity of defective adenoviruses

Inactive Publication Date: 2003-06-04
SHANGHAI SUNWAY BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, this requires that the replication-defective adenovirus used to destroy a specific targe...

Method used

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  • Gene engineering adenovirus and it application
  • Gene engineering adenovirus and it application
  • Gene engineering adenovirus and it application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of Plasmids

[0025] pXC-1 and pBHG11 were purchased from Microbix Biosystems. PXC-1 contains human adenovirus type 5 (Ad5) gene sequence from bp22 to 5790. pBHG11 contains the Ad5 gene sequence with two deletions: the deletion of the E1 region, from bp188 to 1339, the packaging signal used to form the viral DNA capsid protein, and the deletion of the E3 region, from bp27865 to 30995. The DNA of pBHG11 is not infectious, but co-transfection of pXC-1 and pBHG11 can generate infectious virus through homologous recombination.

[0026]In order to construct the subcloning of the Ad5 DNA fragment from bp1338 to bp2501 on pXC-1, the following two synthetic oligonucleotide fragments were used to amplify the PCR product from pXC-1, and the primers were as follows: HZ1 (5'-CTATCCTGAGACGCCCGAC- 3')HZ2(5'-GATCGGATCC AGGTCT CCAGTAAGTGGTAGCTGC-3') (the BgII site is underlined) and cloned into the pGEM-T vector (Promega), resulting in HZ102. To...

Embodiment 2

[0034] Example 2 In Vitro Detection Using Plaque Method

[0035] Firstly, the plaque method was used to detect the growth ability of such viruses in p53-deficient cells, that is, to quantitatively detect the ability of a specific virus to infect a certain cell line. Plaque method is applied to the following cell lines: ovarian cancer cell line (OVCAR-3, p53 mutant strain), liver cancer cell line (Hep3B, p53 mutant strain), glioma cell line (U373, p53 mutant strain), colon cancer cell line Strains (SW620, p53 mutant strain and RKO, p53 normal strain), breast normal cells (HBL-100, p53 normal strain). Because HYH cells express E1A and E1B proteins, S98 virus can efficiently form plaques against these cell lines.

[0036] Table 1 shows the average percentage of replicated samples of the tested viruses. For all cell lines, the titer of a particular virus was normalized to its titer in HYH cells so that the virus was comparable to the corresponding cell line. When the ...

Embodiment 3

[0045] Embodiment 3 in vitro cell test

[0046] The second experiment is to observe the characteristics of different virus replication and the lesions produced by cells. The experimental process is as follows. Infect cells with increasing amounts of viral infection and monitor cell lesions. When it was observed that the MOI was 0.01, the wild-type adenovirus could completely lyse the cultured monolayer cells, which was the end point of the experiment. Human microtubule endothelial cells (hMVEC, derived from human lung), a primary non-renewable cell, were selected to detect its sensitivity to S98 genetically engineered virus and wild-type adenovirus in vitro.

[0047] The results showed that when the MOI of S98-100 was 0.01, the cultured monolayer cells could be completely lysed within 10 days. In contrast, normal monolayer cells infected with S98-002 showed no obvious cytopathic changes at the same time points at MOIs of 10, 1.0, 0.1, and 0.01, respectively. T...

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Abstract

The present invention construts two kinds of gene engineering adenovirus capable of being replicated selectively in human tumor cells with P53 function deficiency for ultimately killing tumor cells. Its tumor resisting curative effect is determined through the human tumor model transplanted to mouse body and test data shows that the gene engineering adenovirus can be used in treating specific tumor.

Description

technical field [0001] The present invention relates to genetically engineered adenoviruses, which can selectively replicate in large quantities in p53 function-deficient tumor cells and eventually kill tumor cells, but they cannot replicate in normal cells. In addition, the present invention also relates to a pharmaceutical composition containing the genetically engineered adenovirus and the use of the genetically engineered adenovirus in preparing a pharmaceutical composition for treating cancer. Background technique [0002] Cancer is an umbrella term for a heterogeneous disease characterized by the unrestricted growth and proliferation of abnormal cells. It is generally believed that the proliferation of normal cells is regulated by two mutually antagonistic genes, namely proto-oncogenes and tumor suppressor genes. Proto-oncogenes promote cell growth, while tumor suppressor genes inhibit cell growth. When the gene is mutated, that is, the proto-oncogene is activated or...

Claims

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Application Information

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IPC IPC(8): A61K39/235A61K45/00C12N7/01
Inventor 张晖
Owner SHANGHAI SUNWAY BIOTECH
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