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CRISPR/Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and lentivirus of CRISPR/Cas9 recombinant lentiviral vector

A technology of recombinant lentivirus and lentiviral vector, applied in the fields of medicine and genetic engineering, can solve the problem of high off-target rate, and achieve the effect of inhibiting the spread of infection and delaying invasion.

Active Publication Date: 2015-04-01
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prevention and treatment of AIDS, CRISPR / Cas9 system has been used to genetically modify CCR5, another co-receptor of HIV (Cradick, Fine et al.2013, Ye, Wang et al.2014), although CD4+CCR5 gene modification of T cells or CD34+ stem cells can prevent HIV-1 virus invasion to a certain extent. The off-target rate of the CRISPR / Cas9 system is very high, and in the acute phase of viral infection, HIV-1 virus mainly uses CCR5 as a co-receptor. Usually (up to 50%) there are amphitropic viruses that can use both CCR5 and CXCR4 as co-receptors in the body, so only knocking out the CCR5 gene has certain limitations on the complete elimination and prevention of AIDS

Method used

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  • CRISPR/Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and lentivirus of CRISPR/Cas9 recombinant lentiviral vector
  • CRISPR/Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and lentivirus of CRISPR/Cas9 recombinant lentiviral vector
  • CRISPR/Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and lentivirus of CRISPR/Cas9 recombinant lentiviral vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] [Example 1] Construction of a CRISPR / Cas9 recombinant lentiviral vector containing a specific target sequence of CXCR4

[0062] 1. Conservative analysis of the target sequence targeting the HIV co-receptor CXCR4 gene

[0063] In order to clarify the conservation of the CXCR4 target sequence selected by the applicant in humans and monkeys, the applicant applied the BLAST software in the National Center For Biotechnology Information Database to analyze the sequence conservation of the CXCR4 gene in humans and rhesus monkeys. In the conserved exon region, select the target sequence that conforms to the characteristics of CRISPR-Cas9 binding genome [5'-G(19N)-NGG3' or 5'-CCN(19N)C-3']. We designed 10 target sites (Table 1), and all target site sequences were completely identical (100%) between humans and monkeys.

[0064] 2. Construction of recombinant lenti-CRISPR and identification of gene editing effects

[0065] The modified lentiviral vector is pLentiCRISPR (Addgene ...

Embodiment 2

[0097] [Example 2] Packaging of CRISPR / Cas9 lentiviral system

[0098] In order to further improve the gene knockout efficiency of the CRISPR / Cas9 recombinant lentiviral vector, the CRISPR / Cas9 recombinant lentiviral vector was packaged into lentivirus in HEK-293T cells.

[0099] The lentiviral packaging vector is:

[0100] lentiCRISPR vectors for different targets

[0101] psPAX2 lentiviral helper packaging plasmid (purchased from Addgene)

[0102] pMD2.G lentiviral helper envelope plasmid (purchased from Addgene)

[0103] The specific process is as follows:

[0104] 1) HEK-293T cells (purchased from CCTCC) were plated in a cell culture dish with a diameter of 10 cm 24 hours before transfection.

[0105] 2) The confluence of the cells should be 80-90% during transfection. The above plasmids were co-transfected into the cells with the standard Lipofectamine2000 transfection reagent, and the medium was changed 6 hours after transfection.

[0106] Transfection system:

[010...

Embodiment 3

[0111] [Example 3] Using CRISPR / Cas9 recombinant lentivirus to establish a cell line that stably knocks out the CXCR4 gene

[0112] 1) Use the lentivirus packaged in Example 2 to infect Ghost CD4 / CXCR4 cells with an m.o.i. of 100 (1ml of virus supernatant added to 4ml of DMEM cells), and after 48 hours, the ratio of CXCR4 positive cells was detected by flow cytometry. Unmodified The PE-CXCR4 positive cell rate of Con cells was 87.1%, while the PE-CXCR4 positive cell rates of lentiCRISPR6# and 7# transformed cells were 23.5% and 29.5%, respectively. Compared with the control, they were down-regulated by 73.02% and 66.1% ( Figure 4 ).

[0113] 2) A small portion of cells were collected for Western blot assay to detect the expression level of CXCR4 ( Figure 5 );

[0114] 3) The rest of the cells were sorted by flow cytometry with PE-CXCR4 antibody, and the purity of the stable cell line was tested after sorting, the results are shown in Figure 6 , subculture the sorted cel...

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Abstract

The invention belongs to the field of pharmaceutical and biological engineering, and relates to a CRISPR / Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and a lentivirus of the CRISPR / Cas9 recombinant lentiviral vector. The recombinant lentiviral vector is prepared by carrying out enzyme digestion on a lentiviral vectorlentiCRISPR by BsmBI and connecting into a BsmBI cohesive end-containing CXCR4 specific target sequence to recombine; the obtained CRISPR / Cas9 recombinant lentiviral vector is capable of mutating gene sequences at four different loci of ahuman immunodeficiency virusco-receptor CXCR4 and themutatuin rate is high and up to 25-75%. The cells transformed by the recombinant lentiviral vector cannot be infected by the human immunodeficiency virus. Compared with theRNAi-Knockdown, ZFN and TALEN technologies, the method has higher efficiency of suppressing the human immunodeficiency virus replication; the system is rapid to construct, simple and low in cost, is capable of preventing the invasion of the human immunodeficiency virus and is suitable for human immunodeficiency virus gene therapy.

Description

technical field [0001] The invention belongs to the fields of medicine and genetic engineering, and more specifically relates to the construction and application of a CRISPR / Cas9 recombinant lentiviral vector system that can be used for AIDS gene therapy. Background technique [0002] Acquired immunodeficiency syndrome (AIDS), caused by HIV infection, is one of the major health threats facing the world. According to the assessment of my country's AIDS jointly released by the Ministry of Health, UNAIDS and the World Health Organization on November 11, 2013, as of August 31, 2013, there were about 428,867 HIV-infected people and patients in China. 127,758 deaths. The AIDS epidemic is severe, and there is still no effective vaccine. Although the current drugs for the treatment of AIDS (such as the "cocktail" therapy HAART) can effectively prolong the survival time of patients, the drug resistance and serious side effects caused by long-term medication make it difficult to cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01A61K48/00A61P31/18
Inventor 郭德银陈述亮侯盼盼陈宇
Owner WUHAN UNIV
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