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IKZF3 gene-silenced T cells and application thereof

A gene silencing and cell technology, applied in tumor immunotherapy and biomedicine, can solve problems such as unclear influence of malignant tumors, and achieve high cell expansion multiples, long-term cell survival, and strong cell killing ability

Inactive Publication Date: 2020-02-11
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The impact of Aiolos dysregulation on human malignancies remains unclear

Method used

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  • IKZF3 gene-silenced T cells and application thereof
  • IKZF3 gene-silenced T cells and application thereof
  • IKZF3 gene-silenced T cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0073] The method for preparing T cells or the method for increasing the activity of T cells of the present invention is to silence the IKZF3 gene of T cells.

[0074] Gene silencing refers to the phenomenon that a specific gene in an organism is not expressed or its expression is reduced.

[0075] Further, antisense RNA, antagomir, siRNA, shRNA, meganuclease (Meganuclease), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) or CRISPR system can be used to target the IKZF3 gene The target sequence silences the IKZF3 gene.

[0076] The target sequence in the IKZF3 gene is shown in SEQ ID NO:1.

[0077] In one embodiment, the aforementioned IKZF3 gene interference nucleic acid molecule, and / or the aforementioned IKZF3 gene interference nucleic acid construct, and / or the aforementioned IKZF3 gene interference lentivirus can be administered to T cells to reduce IKZF3 in T cells gene expression. Thereby preparing the T cells or improving the activ...

Embodiment 1

[0107] This example provides the design and synthesis of sgRNA for specifically targeting the IKZF3 gene in the CRISPR / Cas9 specific knockout of the human IKZF3 gene

[0108] 1. Design of sgRNA targeting human IKZF3 gene

[0109] (1) Select the sequence of 5'-N(21)GG on the IKZF3 gene.

[0110] (2) The targeting site of the sgRNA on the IKZF3 gene is located in the exon of the gene, which is more likely to cause the deletion of the fragment or the frame-shift mutation, so as to achieve the purpose of complete gene inactivation.

[0111] (3) The targeting site of sgRNA on IKZF3 gene is located on the common exon of different splicers.

[0112] (4) Use BLAT in the UCSC database or BLAST in the NCBI database to determine whether the target sequence of the sgRNA is unique and reduce potential off-target sites.

[0113] Based on the above principles, professionals in the field can design several sgRNAs targeting the human IKZF3 gene.

[0114] 2. Selection of sgRNA targeting huma...

Embodiment 2

[0131] Use CRISPR / Cas9 to specifically knock out the human IKZF3 gene (the sgRNA used to target the IKZF3 gene is shown in the sequence table as SEQ ID NO.2)

[0132] 1. The linearized sequence is the pGL3-U6-sgRNA plasmid shown in the sequence table SEQ ID NO.11.

[0133] Enzyme digestion system and conditions are as follows:

[0134] 2 μg pGL3-U6-sgRNA

[0135] 1μl CutSmart Buffer

[0136] 1 μl BsaI (NEB, R0535V)

[0137] Replenish water to 50ul, incubate at 37°C for 3-4 hours, shake and centrifuge at regular intervals to prevent droplets from evaporating onto the tube cap. After the enzyme digestion is completed, use illustra GFX PCR DNA and Gel Band Purification Kits (GE, 28903470) to purify and recover to 20-50 μl sterilized water.

[0138] 2. The double-stranded sgRNA oligonucleotide (its Forward oligo and Reverse oligo sequences are respectively shown in sequence table SEQ ID NO.5, 6) obtained after denaturation and annealing can be connected into the U6 eukaryotic ...

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Abstract

The invention provides a nucleic acid molecule interfered by an IKZF3 gene. The target sequence in the IKZF3 gene is shown as SEQ ID NO: 1, the nucleic acid molecule is sgRNA, and the sequence of thenucleic acid molecule is selected from any one or more of SEQ ID NO: 2-4. The invention also provides T cells, and the IKZF3 gene of the T cells is silenced. The invention provides a method for enhancing the function of engineered T cells and a cell drug obtained by the method. By using the method, the engineered T cells can secrete more cell factors, the engineered T cells have stronger cell killing capability, and the engineered T cells have higher cell expansion times and longer cell survival time.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to the field of tumor immunotherapy, and relates to an IKZF3 gene silenced T cell and its application. Background technique [0002] CAR-T cell therapy is an immunotherapy that uses T cells genetically modified to express a chimeric antigen receptor (CAR) to eliminate tumor cells. CAR is the core component of CAR-T cells, which consists of antigen binding domain (usually scFv derived from antibody), hinge and transmembrane region, intracellular signal region of T cell receptor and intracellular signal region of co-stimulatory receptor Composed in tandem, single-chain antibody scFv can specifically bind tumor-associated antigens, the transmembrane region and intracellular signaling domain can help T cells activate, and co-stimulatory signals can maintain T cell proliferation. [0003] Current research and clinical trials have shown that CAR-T cells have achieved excellent results in the t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N7/01C12N15/867C12N15/90C12N5/10A61K35/17A61P35/00
CPCC12N15/113C12N7/00C12N15/86C12N15/907C12N5/0636C07K14/4702A61K35/17A61P35/00C12N2740/15021C12N2740/15043C12N2510/00C12N2310/20
Inventor 朱学锴邹晏
Owner SHANGHAI TECH UNIV
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