SgRNA combined with immunogene to inhibit high risk HPV expression, gene knockout vector thereof, and application of sgRNA and vector

A technology of gene suppression and gene knockout, applied in the direction of DNA/RNA fragments, carriers, nucleic acid carriers, etc., can solve the problems that drugs cannot effectively clear intracellular infection, few therapeutic drugs, and limited effects, etc., and achieve the purpose of inhibiting tumor growth, The method has simple steps and high knockout efficiency

Active Publication Date: 2016-08-03
李旭
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Problems solved by technology

[0006] There are problems in the eradication of high-risk HPV infection in normal human cervical epithelium and the treatment of advanced cervical cancer: (1) Drugs cannot effectively eliminate high-risk HPV infected in cells; (2) The efficiency of other methods to specifically knock out HPVE6 and E7 genes

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  • SgRNA combined with immunogene to inhibit high risk HPV expression, gene knockout vector thereof, and application of sgRNA and vector
  • SgRNA combined with immunogene to inhibit high risk HPV expression, gene knockout vector thereof, and application of sgRNA and vector
  • SgRNA combined with immunogene to inhibit high risk HPV expression, gene knockout vector thereof, and application of sgRNA and vector

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[0030] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0031] see figure 1 , The directional recognition and cleavage of genes by the CRISPR / Cas9 system are realized by sgRNA and Cas9. sgRNA determines the targeting of Cas9 and also determines the cleavage activity of Cas9. The present invention aims to apply CRISPR / Cas9 technology, take high-risk HPV16+ human cervical cancer cells and HPV16+ xenografted tumor mouse models as research objects, use the target pathogenic gene HPV16, and cooperatively target tumor immune regulation molecule PD-1 Gene editing strategies. Firstly, the gRNA sequence targeting the HPV16 gene was screened in vivo and in vitro to achieve effective knockout of the HPV16 gene; then, the gRNA sequence targeting PD-1 was screened in vivo and in vitro, and the multi-gene co-editing effect was achieved through co-transfection, and then joint intervention was investigated Whether the t...

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Abstract

The invention provides an sgRNA combined with immunogene to inhibit high risk HPV expression, a gene knockout vector thereof, and an application of the sgRNA and the vector. The sgRNA sequences of a human papilloma virus 16 type gene suitable for CRISPR-Cas9 targeting editing and a human PD-1 gene are selected, and the plasmid vector of the sgRNA and a CRISPR-Cas9 nuclease gene expression vector are transferred to HPV16+ human cervical carcinoma cells and HPV16+ transplantation tumor-bearing mice, and can obviously inhibit HPV16 expression and inhibit tumor growth. The gene expression vector prepared in the invention has the advantages of simple method steps, good sgRNA targeting property, high CRISPR-Cas9 knockout efficiency, accurate targeting splicing of high risk HPV16 type and PD-1 genes, efficient reduction of the expression of the high risk HPV16 type gene, and obvious inhibition of the tumor growth through combined application.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biomedicine, and relates to a design method for CRISPR / Cas9 to specifically modify multiple gene targets of high-risk HPV16 and PD-1, and to prevent human cervical cancer by knocking out the oncogene expression of high-risk HPV16. And the combined immune gene therapy strategy was used to enhance the oncogene targeting splicing of high-risk HPV16 to treat xenograft tumors in HPV16+ mice. Background technique [0002] In recent years, genome editing tools have been widely used in the field of biomedicine, and clustered regularly interspaced short palindromic repeats (CRISPR) technology has become a hot spot in genome editing. CRISPR is a sequence naturally present in bacterial DNA that binds to CRISPR-associated (Cas) nucleases that function as guide RNAs to protect the bacterial genome from attack by target-specific sequences detected in invasive phages. CRISPR / Cas9 technology was rated as o...

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Application Information

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IPC IPC(8): C12N15/113C12N15/85A61K31/7088A61K48/00A61P35/00A61P31/20
CPCC07K14/70521C12N15/1131C12N15/1138C12N15/85C12N2310/10C12N2800/107C12N2800/80C12N2810/10
Inventor 甄帅李旭赵乐罗文娟
Owner 李旭
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