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CCR5 deleted hematopoietic stem cell, preparation method and application thereof

A technology of hematopoietic stem cells and pluripotent stem cells, applied in the biological field

Inactive Publication Date: 2011-11-09
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI +1
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  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the traditional gene therapy based on hematopoietic stem cells and T lymphocytes cannot overcome the bottleneck of low transfection efficiency and inability to effectively expand hematopoietic stem cells in vitro

Method used

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  • CCR5 deleted hematopoietic stem cell, preparation method and application thereof
  • CCR5 deleted hematopoietic stem cell, preparation method and application thereof
  • CCR5 deleted hematopoietic stem cell, preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0048] Knock out the CCR5 gene in iPS cells and induce differentiation into CCR5-deficient hematopoietic stem cells in vitro.

[0049] Construction of CCR5 gene knockout-related vectors ( figure 1 ):

[0050] 1. Construction of CCR5-specific zinc finger nuclease expression vector. Based on the pXL-Bac II (gifted by Malcolm J. Fraser, University of Notre Dame) vector, the CAG promoter (references: Alexopoulou A, CouchmanJ, Whiteford J. The CMV early enhancer / chicken βactin (CAG) promoter can be used to drivetransgene expression during the differentiation of murine embryonic stem cells into vascular progenitors. BMC Cell Biology. 2008; 9: 2) and cloned into the vector with Sal I and BamH I to obtain the pXG vector. 再将合成的一对CCR5特异性锌指核酸酶基因(锌指核酸酶1基因:ATGGACTACAAGGACCACGATGGCGATTACAAGGACCACGACATTGATTACAAGGACGATGATGACAAGATGGCACCAAAAAAGAAACGAAAAGTGGGCATCCACGGGGTCCCAGCCGCTATGGCAGAACGACCCTTTCAGTGCCGCATCTGTATGCGAAACTTCAGCGACAGATCCAACCTGTCTAGGCACATTCGCACTCATACCGGCGAGAAACCATTTGCTTGCGATATCTG...

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Abstract

The invention discloses a method for preparing auto CCR5 deleted hematopoietic stem cells. The method comprises the steps that: CCR5 specific zinc finger nucleases (ZFNs) expression plasmids, and enhanced green fluorescent protein (EGFP) and puromycin recombinant plasmids are cotransfected into human induced pluripotent stem cells; human induced pluripotent stem cell CCR5 is specifically inactivated through gene insertion; and in vitro directional induction and differentiation are carried out, such that CCR5 deleted hematopoietic stem cells are obtained. The hematopoietic stem cells provided by the invention can be applied in the treatment of AIDS. With the hematopoietic stem cells provided by the invention, a novel method for treating AIDS stem cells can be provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an autologous CCR5-deficient hematopoietic stem cell and its preparation method and application. Background technique [0002] AIDS (AIDS) is a serious infectious disease that threatens all mankind. So far, humans have not invented a cure for AIDS, nor have they invented an effective AIDS preventive vaccine. Highly effective antiretroviral therapy (HARRT, commonly known as "cocktail" therapy) can inhibit the replication of HIV (HIV), effectively alleviate the disease, and significantly reduce the mortality rate, but it cannot completely remove the virus in the body, and the virus will rebound quickly after stopping the drug. Therefore, the purpose of radical cure cannot be achieved. Through the study of the process of HIV infecting cells, it is known that HIV enters cells and replicates in cells through the interaction with cell membrane molecules such as T cell membrane receptor C...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N15/63C12N15/09A61K35/12A61P31/18
Inventor 陈小平姚永超秦莉那顺巴雅尔赵思婷覃丽梅何正祥
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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