Conditional replicative adenoviral vector and application of viral replication regulated by transcriptional repressive tet-on system

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

Active Publication Date: 2020-11-06
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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  • Conditional replicative adenoviral vector and application of viral replication regulated by transcriptional repressive tet-on system
  • Conditional replicative adenoviral vector and application of viral replication regulated by transcriptional repressive tet-on system
  • Conditional replicative adenoviral vector and application of viral replication regulated by transcriptional repressive tet-on system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Conditionally it is replicating adenoviral vectors according to the present embodiment constructed virus replication by transcription Tet-on system suppressor TetR-KRAB regulation mediated follows:

[0031] Adenovirus E1A-E1B19KD adenovirus E1 region comprises elements for the connection sequence 5'-CMV promoter, TetR-KRAB gene, tetracycline responsive promoter element TRE3G-E1b Pro, EcoRⅠ restriction sites, deletions of the protein E1BΔ55KD sequences, restriction sites SpeⅠ, mRNA transcription termination signals SV40polyA-3 '; CMV promoter expression transcriptional repressor TetR-KRAB; EcoRⅠ two restriction sites SpeI and inserted into the gene sequence for translation start site of the adenovirus E1A point adenovirus E1B 19KD protein gene stop codon fragment, containing the tetracycline responsive promoter element TRE3G-E1b Pro adenovirus expressing E1A gene, the adenovirus E1B 19KD gene by the expression of its own promoter, adenovirus obtained after packaging named Ad...

Embodiment 2

[0057] Conditionally replicating adenovirus viral replication by inhibiting the transcriptional regulation of type Tet-on system TetR-KRAB vector-mediated infection of human cervical carcinoma Hela cells in the regulation of replication

[0058] Same conditions replicative adenovirus vector virus replication according to the present embodiment constructed by the transcriptional repressor Tet-on system type TetR-KRAB regulation mediated described in Example 1.

[0059] Which steps are as follows:

[0060] Example 1-4 Step this embodiment is identical to Example 1.

[0061] Conditionally replicating adenovirus 5, inhibition of viral replication under the transcriptional regulation of type Tet-on system TetR-KRAB vector-mediated infection of Hela cells

[0062] Hela cells, after the mass fraction of 0.1% trypsin, by 5 × 10 4 / Well plated on 24-well plate, and 600μL was added NCS containing 10% DMEM culture overnight. Every other day of infection, the virus Ad5-CMV-TetR-KRAB-TRE3G-E1...

Embodiment 3

[0094] Replication regulation U87MG tumor cells after conditionally replicative adenovirus viral replication by inhibiting the transcriptional regulation of type Tet-on system TetR-KRAB vector-mediated infection of human malignant glioblastoma brain

[0095] Same conditions replicative adenovirus vector virus replication according to the present embodiment constructed by the transcriptional repressor Tet-on system type TetR-KRAB regulation mediated described in Example 1.

[0096] Which steps are as follows:

[0097] Example 1-4 Step this embodiment is identical to Example 1.

[0098] Cells used in method step 5-7 which is replaced by Hela cells in Example 2 of U87MG cells, remaining the same manner as in Example 2.

[0099] Viral genome replication see test results Figure 4The results show, conditionally replicating adenovirus embodiment virus present embodiment replication by TetR-KRAB mediated transcriptional suppression type Tet-on system regulated Ad5-CMV-TetR-KRAB-TRE3G-E1bP...

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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 present invention relates to adenoviral vector and its application in the field of life science, specifically to a viral replication by transcription-inhibiting conditions Tet-on system regulated TetR-KRAB-mediated replication of adenoviral vectors and their use. Background technique [0002] Tumor is the most important disease threat to human health. Despite the widespread application of traditional treatment, but because of the lack of tumor specificity, often affect normal cells cause greater side effects. Conditionally replicating adenovirus (CRAd), a new type of virus is based on evolved adenovirus for cancer gene therapy, the virus can be specifically amplified in tumor cells, but does not replicate in normal cells or copy is weak, it is also known oncolytic adenovirus (oncolyticAdenovirus). CRAd currently three main types: 55KD deletion (1) Ad5E1B region Ad5E1B △ 55KD (such as ONYX-015), which can replicate in a tumor cell abnormal p53 function, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861
CPCC12N15/86C12N2710/10043C12N2830/006
Inventor 夏海滨陈皓
Owner SHAANXI NORMAL UNIV
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