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Tumor-targeting gene therapy drug based on CRISPR/Cas9 gene editing technology and use thereof

A gene and targeting technology, applied in the field of tumor-targeting gene therapy drugs based on CRISPR/Cas9 gene editing technology and its application, can solve the problem of targeted knockout of tumor cell DNMT1 that is difficult to achieve therapeutic effect and cannot be effective, durable and stable Gene and other problems to achieve the effect of inhibiting the growth of tumor cells

Pending Publication Date: 2019-07-05
CHENGDU JINKAI BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Chinese patent document CN104971361A discloses CRISPR / Cas9 technology targeted editing of human DNMT1 gene and its use in the treatment of tumors, but the sgRNA involved and the sgRNA loaded into the plasmid vector cannot effectively and persistently target Knocking out the DNMT1 gene in tumor cells makes it difficult to achieve a stable therapeutic effect

Method used

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  • Tumor-targeting gene therapy drug based on CRISPR/Cas9 gene editing technology and use thereof
  • Tumor-targeting gene therapy drug based on CRISPR/Cas9 gene editing technology and use thereof
  • Tumor-targeting gene therapy drug based on CRISPR/Cas9 gene editing technology and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Design of CRISPR-Cas9 to knock out sgRNA specifically targeting DNMT1 gene in human DNMT1 gene

[0067] 1. Select 5'-GN(18-21)GG on the human DNMT1 gene, where 3'-NGG is PAM. Therefore, the length of the sgRNA targeting the human DNMT1 gene is 18-21 bp.

[0068] 2. When designing sgRNA, use G as the starting base at the 5'end.

[0069] 3. The target binding region of sgRNA on the human DNMT1 gene is the conserved domain methyltransferase domain of the gene, that is, exons 31 and 32 of the human DNMT1 genome.

[0070] 4. Using online software (http: / / zifit.partners.org / ZiFiT / ) and supplemented by manual inspection, a total of 35 sgRNAs targeting exons 31 and 32 of the human DNMT1 genome were designed, which have targets The base sequence of the sgRNA that knocked out the human DNMT1 gene is shown in SEQ ID NO.: 1-7.

Embodiment 2

[0071] Example 2. Construction of sgRNA plasmid vector targeting human DNMT1 gene:

[0072] 1. Preparation of linear PX330 plasmid vector:

[0073] The reaction system is as follows:

[0074] PX330 plasmid vector: 3μg

[0075] BbsⅠ enzyme: 2μl

[0076] Buffer: 2μl

[0077] Double distilled water volume: 20μl

[0078] In the reaction system, the PX330 plasmid vector was digested with BbsI overnight to form a linear PX330 plasmid vector. The same method can be used to prepare linear PX459 granulation carrier.

[0079] 2. Design and double-stranded preparation of sgRNA oligonucleotide targeting human DNMT1 gene

[0080] (1) Select two of the sgRNAs designed in Example 1 (as shown in SEQ ID NO: 1 or 4 in the sequence table, respectively). According to the Bbs I digestion method used in the plasmid vector of step 1, in the selected Add the CACC linker to the 5'ends of the two sgRNAs to obtain the forward oligo; according to the selected sgRNA, obtain the complementary strand, and add the linke...

Embodiment 3

[0108] Example 3: Nci1 restriction enzyme digestion and Sanger sequencing method verify the sgRNA / Cas9-mediated targeted cleavage of the DNMT1 gene in SKOV3 cells

[0109] 1. SKOV3 cell plating culture:

[0110] The SKOV3 cells in the logarithmic growth phase were prepared as a cell suspension, and diluted with 10% FBS-1640 to 5×10 4 cells / ml, inoculate 100μl / well to a 96-well cell culture plate, 37.0℃, 5% CO 2 Cultivate for 24h under conditions. After the cells adhered, they were replaced with serum-free 1640 medium and starved for 24 hours.

[0111] 2. Liposome transfection method:

[0112] The PX459-DNMT1-1 plasmid (sgRNA sequence: SEQ ID NO: 1) constructed in Example 2 was transfected into the SKOV3 cells cultured in step 1 using the liposome transfection method (such as transfection reagent lipo2000), In the control group, SKOV3 cells were transfected with PX459 empty plasmid vector and incubated at 37°C for 24h.

[0113] 3. Screen positive cells with puromycin, collect the cells...

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Abstract

The present invention provides an sgRNA specifically targeting a human DNMT1 gene based on a CRISPR / Cas9 system, a use thereof for guiding the CRISPR / Cas9 system in a drug for treatment and / or prevention of tumors, and a method correspondingly constructing a sgRNA expression vector. The CRISPR / Cas9 system can rapidly, efficiently and specifically knock out the human DNMT1 gene, induces inactivation of the DNMT1 gene in tumor cells, inhibits expression of the DNMT1 and methylation of DNA and thus inhibits tumor cell growth.

Description

Technical field [0001] The present invention belongs to the technical field of molecular biology and biomedicine, and in particular relates to a CRISPR / Cas9 system-based sgRNA that specifically targets human DNMT1 gene, and its CRISPR / Cas9 vector, and the CRISPR / Cas9 specifically targeted knockout of DNMT1 gene The Cas9 system is used in the preparation of tumor gene therapy drugs. Background technique [0002] Malignant tumors are currently one of the main diseases endangering human health. According to the latest cancer statistics, there are 17 million new cancer cases worldwide and 9 million deaths every year; in 2015, the number of new cancer cases and deaths in my country was 4.292 million and 281.4 cases, respectively. Shows a clear trend of younger people. Although in the past 20-30 years, traditional treatment methods including radiotherapy, chemotherapy and surgical treatment have been continuously developed and have achieved certain effects in the treatment of cancer, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22A61K31/7105A61K48/00A61P35/00
CPCC12N15/113C12N15/907C12N9/22A61K31/7105C12N2310/10
Inventor 姚少华魏于全何志尧彭飞赵志伟夏言富
Owner CHENGDU JINKAI BIOTECH CO LTD
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