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Conditionally replicating adenovirus vector for viral replication regulated by transcription inhibition type Tet-On system and application

A virus replication and tet-on technology, applied in the field of life sciences, can solve the problems of ineffective delivery of viruses, cracking, loss of vitality, etc., and achieve the effect of increasing the virus load

Active Publication Date: 2017-11-10
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the viral load is too high, hMSCs may lose their vitality or even be lysed due to viral replication, resulting in the inability to effectively deliver the virus to the tumor tissue

Method used

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  • Conditionally replicating adenovirus vector for viral replication regulated by transcription inhibition type Tet-On system and application
  • Conditionally replicating adenovirus vector for viral replication regulated by transcription inhibition type Tet-On system and application
  • Conditionally replicating adenovirus vector for viral replication regulated by transcription inhibition type Tet-On system and application

Examples

Experimental program
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Effect test

Embodiment 1

[0030] The viral replication constructed in this example is regulated by the TetR-KRAB-mediated transcriptional repressive Tet-on system, and the conditional replicative adenoviral vector is as follows:

[0031] The E1 region of adenovirus contains elements and the connecting sequence is 5'-CMV promoter, TetR-KRAB gene, promoter TRE3G-E1b Pro containing tetracycline response element, EcoR Ⅰ restriction site, adenovirus E1A- E1B19KD gene sequence, restriction site Spe Ⅰ, mRNA transcription termination signal SV40polyA-3'; CMV promoter expresses transcriptional repressor TetR-KRAB; the gene sequence inserted in the two restriction sites EcoR Ⅰ and Spe Ⅰ is adenovirus The E1A translation start site to the adenovirus E1B 19KD protein stop codon gene segment, the promoter TRE3G-E1b Pro containing the tetracycline response element expresses the adenovirus E1A gene, and the adenovirus E1B 19KD gene is expressed by its own promoter, after The packaged adenovirus was named Ad5-CMV-TetR...

Embodiment 2

[0056] Viral replication regulated by TetR-KRAB-mediated transcriptional repression Tet-on system Replication regulation of conditionally replicable adenoviral vectors infected with human cervical cancer HeLa cells

[0057] The conditional replicative adenoviral vector constructed in this example, whose viral replication is regulated by the TetR-KRAB-mediated transcriptional repressive Tet-on system, is the same as that in Example 1.

[0058] Its method steps are as follows:

[0059] Steps 1-4 of the method in this embodiment are exactly the same as in Example 1.

[0060] 5. Viral replication is regulated by the TetR-KRAB-mediated transcriptional repressive Tet-on system and the conditional replicative adenoviral vector infects Hela cells

[0061] Hela cells, after digested with 0.1% trypsin, weighed 5×10 4 Spread on a 24-well plate per well, and add 600 μL of DMEM containing 10% NCS to culture overnight. The infection was carried out every other day, and the virus Ad5-CMV-...

Embodiment 3

[0092] Viral replication is regulated by the TetR-KRAB-mediated transcriptional repressive Tet-on system. Replication regulation after infection of human malignant glioblastoma cell U87MG by a conditionally replicable adenoviral vector

[0093] The conditional replicative adenoviral vector constructed in this example, whose viral replication is regulated by the TetR-KRAB-mediated transcriptional repressive Tet-on system, is the same as that in Example 1.

[0094] Its method steps are as follows:

[0095] Steps 1-4 of the method in this embodiment are exactly the same as in Example 1.

[0096] The cells used in steps 5-7 of the method were replaced by U87MG cells from Hela cells in Example 2, and the remaining methods were the same as in Example 2.

[0097] The results of detection of viral genome replication can be found in Figure 4, the results showed that the conditional replication adenovirus Ad5-CMV-TetR-KRAB-TRE3G-E1b Pro-Δ55KD infecting U87MG cells was regulated by th...

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Abstract

The invention discloses a conditionally replicating adenovirus vector for virus replication regulated by a TetR-KRAB-mediated transcription inhibition type Tet-On system, adenovirus E1 region comprises the following components in the following connection sequence: 5'-CMV promoter, TetR-KRAB gene, promoter TRE3G-E1b Pro containing a tetracycline responsive element, EcoR I enzyme cutting site, E1B Delta 55KD protein-deleted adenovirus E1A-E1B19KD gene sequence, enzyme cutting site Spe I and mRNA transcription termination signal SV40polyA-3'; the CMV promoter expresses transcription inhibitor TetR-KRAB; a gene sequence inserted into the two enzyme cutting sites of the EcoR I and the Spe I is a gene fragment from adenovirus E1A translation initiation site to adenovirus E1B19KD protein termination codon, the promoter TRE3G-E1b Pro containing the tetracycline responsive element expresses adenovirus E1A gene, adenovirus E1B19KD gene is expressed by the own promoter of the adenovirus E1B19KD, and the packed adenovirus is named as Ad5-CMV-TetR-KRAB-TRE3G-E1bPro-Delta 55KD. Tests show that the virus can be loaded into mesenchymal stem cells for application in study of tumor therapy.

Description

technical field [0001] The invention relates to an adenovirus vector in the field of life sciences and an application thereof, in particular to a conditional replication adenovirus vector and an application thereof in which virus replication is regulated by a TetR-KRAB-mediated transcriptional repression Tet-on system. Background technique [0002] Tumor is the most important disease that threatens human health. Although traditional treatment methods are widely used, they often cause large toxic side effects on normal cells due to the lack of tumor specificity. Conditionally replicating adenovirus (CRAd) is a new type of virus developed on the basis of adenovirus for tumor gene therapy. The virus can specifically amplify in tumor cells, but does not replicate in normal cells. , or the replication is weak, so it is also called oncolytic adenovirus (oncolytic Adenovirus). At present, CRAds are mainly divided into three categories: (1) Ad5 E1B Δ55KD (such as ONYX-015) with 55...

Claims

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Application Information

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IPC IPC(8): C12N15/861
CPCC12N15/86C12N2710/10043C12N2830/006
Inventor 夏海滨陈皓
Owner SHAANXI NORMAL UNIV
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