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Expression vector for targeted inhibition of HIV-1 provirus expression as well as preparation method and application of expression vector

An HIV-1 and expression vector technology, applied in the field of bioengineering, can solve hidden dangers, HIV-1 integrase off-target safety and other problems, and achieve the effect of inhibiting virus replication

Active Publication Date: 2018-06-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2013, Martinez-Colom A et al. constructed a chimeric protein expression vector containing the HIV-1 integrase domain targeting the HIV-1 regulatory region LTR (Long terminal repeat) and the catalytic domain of DNA methyltransferase Dnmt3b , found that the chimeric protein can cause the methylation of the HIV-1 regulatory region and lead to the inhibition of HIV-1 expression, but HIV-1 integrase has a potential safety hazard of "off-target"
However, there have been no reports or patents on HIV-1 proviral expression silencing mediated by the gene editing system of chimeric DNA methyltransferase

Method used

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  • Expression vector for targeted inhibition of HIV-1 provirus expression as well as preparation method and application of expression vector
  • Expression vector for targeted inhibition of HIV-1 provirus expression as well as preparation method and application of expression vector
  • Expression vector for targeted inhibition of HIV-1 provirus expression as well as preparation method and application of expression vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Screening of an effective chimeric DNA methyltransferase ZF-Dnmt1 by an in vitro dual-luciferase reporter gene detection system: human embryonic kidney cells HEK293 cells were used as target cells, and the recombinant plasmid ZF-Dnmt1 containing zinc finger structure DNA methyltransferase was constructed -Dnmt1, pcDNA3.1-Dnmt1 or pcDNA3.1-ZF2 empty vectors were co-transfected with LTR-regulated luciferase plasmid pGL3-LTR-luc and internal reference gene expression plasmid pRL-SV40 into HEK-293T cells, and detected after 72h The relative activity of luciferase, its value can reflect the effect of ZF-Dnmt1 on luciferase activity, the results show that the four methylases with chimeric zinc finger structure all silence the expression of luciferase gene The silencing efficiency of ZF2-Dnmt1 and ZF3-Dnmt1 is about 80%; the silencing efficiency of pcDNA3.1-Dnmt1 transfection group is about 30% (such as figure 2 shown in 74).

Embodiment 2

[0030] Detection of the regulation of LTR in TZM-bl cells by DNA methyltransferase with chimeric zinc finger structure: TZM-bl cells were used as test cells, under the premise of transfecting or not transfecting Tat expression plasmid pCMV-Tat, ZF2 -Dnmt1, ZF3-Dnmt1 and pcDNA3.1 empty vectors were transfected into TZM-bl cells, and after 72 hours, the expression level of luciferase was detected, because the expression of luciferase in TZM-bl cells was Tat-dependent , Therefore, in this embodiment, when the Tat expression plasmid exists, the influence of ZF2-Dnmt1 and ZF3-Dnmt1 on the expression of luciferase is mainly detected. The results show that, compared with the pcDNA3.1 transfection group, the ZF2-Dnmt1 transfection group The expression of the luciferase gene can be significantly silenced, and the silencing efficiency is about 60%; while the silencing effect is not seen in the ZF3-Dnmt1 transfection group (such as image 3 shown).

Embodiment 3

[0032] Silencing efficiency and time detection of DNA methyltransferase with chimeric zinc finger structure in virus-infected cell model (YA): YA cells were used as test cells, and ZF2-Dnmt1 or pcDNA3.1 empty vectors were transfected into YA cells by electroporation After three days, the change of the proportion of GFP-positive cells was detected by flow cytometry. The results showed that, compared with the pcDNA3.1 control group, the proportion of GFP-positive cells in the ZF2-Dnmt1 transfection group decreased significantly, and the decline rate was about 25%. (Such as Figure 4 shown); Afterwards, the YA cells transfected with ZF2-Dnmt1 were subcultured, and the cells were harvested at 5 days, 10 days and 15 days after transfection, and the changes in the proportion of GFP-positive cells were detected by flow cytometry, and the results showed that the HIV-1 proviral silencing effect within the ZF2-Dnmt1 transfection group persisted between different generations (e.g. Figu...

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Abstract

The invention belongs to the field of gene therapy and relates to preparation and an application of an expression vector for targeted inhibition of HIV-1 provirus expression by use of embedded DNA methyltransferase adopting a zinc finger structure. The expression vector carrying the DNA methyltransferase adopting the zinc finger structure is proved to be able to inhibit silence of HIV-1 provirus expression in HIV-1 infection and latent infection cell models, the vector can effectively inhibit virus replication in virus infected cells and latent infected cells for 15-40 days or above. Besides,provirus expression silence caused by the expression vector carrying the embedded methyltransferase adopting the zinc finger structure can be inhered among cell generations and has certain safety in cells. A new idea is provided for curing AIDS.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to an expression vector for targeting and inhibiting the expression of HIV-1 provirus and its preparation method and application, in particular to a DNA with a chimeric zinc finger structure for targeting and inhibiting the expression of HIV-1 provirus Preparation and application of expression vector of methyltransferase. Background technique [0002] Studies have shown that the main reason why AIDS (Acquired immunodeficiency virus, AIDS) cannot be completely cured is that HIV-1 hides in the immune cells it infects, and can undergo gene silencing through epigenetic and other mechanisms, thereby evading the immune system and antiretroviral drugs. However, the study also showed that the gene silencing of the HIV-1 provirus in these latently infected cells is reversible, and once the appropriate conditions are encountered, the virus can quickly resume its replication and transcription capa...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/62A61K48/00A61P31/18
CPCA61K48/0008A61K48/005C07K2319/81C12N9/1007C12Y201/01
Inventor 朱焕章邓俊骁季海燕朱豫琪
Owner FUDAN UNIV
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