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Method for knocking out PD-1 gene by utilizing CRISPR/Cas9 technology to construct MSLN-targeted novel CAR-T cell and application of method

A PD-1 and gene technology, applied in the fields of genetic engineering, immunology and oncology, can solve the problems of no observed side effects, increased white blood cell level, etc., and achieve the effect of simple steps and high killing rate

Pending Publication Date: 2017-03-08
NANJING KAEDI BIOTECH INC
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Problems solved by technology

In this Phase 1 clinical study, although five patients experienced side effects such as sepsis, shortness of breath and elevated white blood cell levels during treatment, the researchers did not observe major side effects

Method used

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  • Method for knocking out PD-1 gene by utilizing CRISPR/Cas9 technology to construct MSLN-targeted novel CAR-T cell and application of method
  • Method for knocking out PD-1 gene by utilizing CRISPR/Cas9 technology to construct MSLN-targeted novel CAR-T cell and application of method
  • Method for knocking out PD-1 gene by utilizing CRISPR/Cas9 technology to construct MSLN-targeted novel CAR-T cell and application of method

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Embodiment

[0050] A method for constructing a novel CAR-T cell targeting MSLN by using CRISPR / Cas9 to specifically knock out the human PD-1 gene, comprising the following steps:

[0051] 1) Construction of viral vectors carrying PD-1 gene sgRNA oligonucleotides

[0052] The target sequence of the sgRNA on the PD-1 gene conforms to the sequence arrangement rules of 5'-N(20)NGG, and the target sequence of the sgRNA on the PD-1 gene is located in the exon of the gene; the target sequence of the sgRNA on the PD-1 gene The target sequence is unique, and the target site sequence of the sgRNA on PD-1 consists of the human PD-1 gene targeting sgRNA sequence PD-1-Tn shown in SEQ ID No.51 to SEQ ID No.56;

[0053] The mRNA sequence of the human PD-1 gene consists of human PD-1 gene exons shown in SEQ ID No.46 to SEQ ID No.50;

[0054] Synthesize sgRNA oligonucleotide double strands (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.), anneal at 95°C for 5 minutes after mixing, and then con...

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Abstract

The invention discloses a method for knocking out a PD-1 gene by utilizing a CRISPR / Cas9 technology to construct an MSLN-targeted novel CAR-T cell. According to the method, a CAR-T cell is synchronously infected with a virus solution carrying sgRNA and Cas9-HF nuclease of a human PD-1 gene by utilizing the CRISPR / Cas9 technology to knock out the human PD-1 gene, so that the MSLN-targeted novel CAR-T cell is obtained. The novel CAR-T cell can be used for preparing a preparation used for treating solid tumors. A preparation method is simple in step, and the obtained CAR-T cell has high killing rate for solid tumor cells.

Description

technical field [0001] The present invention relates to the fields of genetic engineering, immunology and oncology, in particular to a method for knocking out the human PD-1 gene using CRISPR / Cas9 technology to construct a new type of CAR-T cell that can target MSLN (Mesothelin, mesothelin) and its application. Background technique [0002] With the deepening of industrialization, air pollution, environmental problems, food safety and changes in people's lifestyles, cancer has become the number one killer threatening human health and the main obstacle to social development in this century. Immune cell biotherapy technology for cancer is an emerging tumor treatment model with significant curative effect. It uses genetic engineering technology to modify, culture and expand immune cells collected from patients in vitro, and then reinfuse them into patients. , to stimulate and enhance the body's own immune function, so as to achieve the purpose of inhibiting tumor growth and tr...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10A61K35/17A61P35/00
CPCC12N15/86A61K35/17C07K14/70521C12N5/0636C12N2510/00C12N2740/15043C12N2800/40
Inventor 代红久李晓亮孙彬张征赵旭东熊波
Owner NANJING KAEDI BIOTECH INC
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