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Zinc finger endonuclease mediating integrated hiv‑1 proviral gene knockout and its preparation and application

A technology of zinc finger nucleic acid and endonuclease, which is applied in applications, gene therapy, antiviral agents, etc.

Active Publication Date: 2017-08-18
SHANGHAI XINHAO BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective means for this problem in this field. Therefore, there is an urgent need in this field to develop a method for directional removal of HIV proviruses on the chromosomes of target cells, so as to fundamentally solve the treatment problem of HIV / AIDS.

Method used

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  • Zinc finger endonuclease mediating integrated hiv‑1 proviral gene knockout and its preparation and application
  • Zinc finger endonuclease mediating integrated hiv‑1 proviral gene knockout and its preparation and application
  • Zinc finger endonuclease mediating integrated hiv‑1 proviral gene knockout and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Identification of zinc finger endonuclease recognition sites in HIV proviruses

[0108] Due to the variability and complexity of HIV-1, it is easy to escape the attack of the immune system, drugs and vaccines, so the identification of zinc finger endonuclease recognition sites is a key step. In this example, the inventors carefully compared 344 HIV-1 subtypes, and finally determined the following sequence as the recognition site sequence:

[0109] CCAGATCTGAGCCTGGGAgctctCTGGCTAACTAGGGA (SEQ ID NO.: 1)

[0110] This sequence is very conserved in HIV-1 5'LTR and 3'LTR. Among the 344 HIV-1 subtypes, the conservation degree of this sequence in HIV-1 5'LTR and 3'LTR is as high as 93%.

Embodiment 2

[0112] Construction of a pair of ZFN-LTR plasmids (pZFN1 plasmid and pZFN2 plasmid)

[0113] In this example, the inventors prepared a pair of pUC plasmids carrying nucleotide sequences encoding zinc finger endonucleases (ZFN1 or ZFN2), that is, pZFN1 that recognizes the site of CCAGATCTGAGCCTGGGA (SEQ ID NO.: 2) and pZFN1 that recognizes pZFN2 at the CTGGCTAACTAGGGA (SEQ ID NO.: 3) site.

[0114] The preparation method of pZFN1 and pZFN2 plasmids is as follows: synthesize 3flag-NLS-zinc finger-FokI oligonucleotide sequence, and introduce NcoI and AvaI restriction sites, oligonucleotide fragments and pZFN at the 5' and 3' ends respectively The backbone plasmids were digested with NcoI and AvaI respectively, ligated, transformed into recipient bacteria, single clones were screened on a KanR plate, the plasmids were extracted, identified by enzyme digestion, and further sequence analysis confirmed.

[0115] figure 1 , figure 2 It is a structural diagram of a pair of ZFN-LTR ...

Embodiment 3

[0119] ZFN-LTR mediates recombinant excision of integrated HIV provirus in HIV-1-infected cells

[0120] Construction and preparation of HIV-1 infected Jurkat cells: HIV-1 pseudovirus pNL4-3-EGFP carrying the EGFP reporter gene (pNL4-3-EGFP was purchased from the National Institutes of Health AIDS Reagent Distribution Center) was used to infect commercially available conventional human T lymphocyte Jurkat strain, GFP positive and negative cell populations were obtained by cell sorting. The GFP-positive cell population was used as a cell model of HIV infection for subsequent experiments.

[0121] The HIV-infected cell model carrying the EGFP reporter gene was cultured and plated, Nucleofector nuclear transfection or liposome transfection of different doses of ZFN-LTR expression plasmids, transfected with ZFN empty plasmids or untransfected cells were used as negative or blank controls, transfected Cells were collected 2, 3, 4, 5, and 6 days after transfection, and flow cytomet...

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Abstract

The invention relates to a mediated integration HIV-1 (Human Immunodeficiency Virus-1) provirus gene knockout zinc finger nuclease as well as a preparation method and an application thereof, provides a target sequence which is highly conserved in each subtype of HIV virus and can be used for specific recognition of the zinc finger nuclease, and also provides a pair of zinc finger nucleases for specific recognition of the target sequence and specific cutting of a corresponding site of the target sequence. Tests prove that the zinc finger nuclease can efficiently and specifically remove HIV-1 proviruses integrated in a host genome and can be used for gene therapy of HIV.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a zinc finger endonuclease mediating the knockout of the integrated HIV-1 provirus gene and its preparation method and application. Background technique [0002] Acquired Immunodeficiency Syndrome (AIDS) is an infectious disease caused by HIV infection that seriously endangers people's life and health. According to WHO statistics, there are more than 50 million AIDS patients in the world, and 5 million new patients are added every year, while the number of people who die from AIDS is about 3 million every year. [0003] At present, the clinical treatment of AIDS is mainly Highly active antiretroviral therapy (HAART), which not only controls HIV replication, but also can rebuild the immune function of AIDS patients, which opens the door of hope for the treatment of AIDS. However, this therapy can only inhibit virus replication and cannot completely eliminate th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A61K38/46A61K48/00A61P31/18
Inventor 朱焕章曲喜英王鹏飞
Owner SHANGHAI XINHAO BIOLOGICAL TECH
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