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Gene biological agent as well as preparation method and application thereof

A gene and gene knockout technology, applied in the field of gene editing and biological cells, can solve the problems of side effects, gene silencing, gene fragment deletion, etc., and achieve the effect of broad application prospects and high gene editing efficiency.

Pending Publication Date: 2022-04-19
SHENZHEN FIRST CONDOR BIOSCIENCE CO LTD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] Under normal circumstances, in the CRISPR-Cas9 gene knockout technology, a gRNA is used to target the target sequence for gene splicing, and the deletion during repair is used to carry out gene splicing, increase bases, cause frameshift mutations, and result in gene silencing; In another case, two gRNAs are used to target the target sequence for gene splicing, resulting in the deletion of gene fragments, resulting in gene silencing
However, there may be a problem in both methods: even if the gene frame shift or gene fragment deletion is caused, the gene can still express the protein normally. At this time, the protein expressed by the gene is not the original wild-type protein, and its function and traits may be altered, and this gene may cause other side effects

Method used

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  • Gene biological agent as well as preparation method and application thereof
  • Gene biological agent as well as preparation method and application thereof
  • Gene biological agent as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] The CAR-T cell structure and cell preparation used for functional verification in this example include the following steps:

[0062] S100: Isolation of peripheral blood PBMCs and culture of T cells

[0063] Mononuclear cells were isolated from donor peripheral blood, density gradient centrifugation was performed using ficol, and T cells were enriched with a T cell sorting kit (CD3 MicroBeads, human-lyophilized, 130-097-043), using conjugated anti-CD3 / anti-CD28 magnetic beads activated culture and expansion of T cells; the medium used TexMACS GMP Medium (MiltenyiBiotec, 170-076-309), containing 10% FBS, 2mM L-glutamine, 100IU / ml rhIL2, all T cells Place at 37°C, 5% CO 2 Cultured in a constant temperature incubator to obtain T cells.

[0064] S200: Cell line culture

[0065] Cell line overexpressing CLDN18.2: 293T (human embryonic kidney cell line), purchased from ATCC.

[0066] Cell line expressing CLDN18.2: 293T-CLDN18.2, constructed by self-produced lentivirus inf...

Embodiment 2

[0082]This example is used to illustrate the effect of inserting the IN044 sequence into the extracellular segment of the CAR structure on the expression of CAR. The specific steps are as follows:

[0083] S500: expression of CAR positive rate in CT106 and CT090 cells.

[0084] Figure 3a , 3b , 3c respectively represent the CAR positive rate detection charts of NT cells, CT106 and CT090. Such as Figure 3a , 3b As shown in , 3c, with NT cells as negative control, CT106 and CT090 cells express green fluorescent protein GFP normally, indicating that the gene expression is normal, and the expression level of GFP can be regarded as the positive rate of gene expression. However, the expression of CAR in CT090 with IN044 insertion sequence was completely silenced, which proved that when the IN044 sequence was inserted into the extracellular region of the membrane-expressed protein, the translated protein of this sequence could mediate protein degradation and realize protein exp...

Embodiment 3

[0093] This example is used to illustrate that the IN044 sequence can silence the expression of PD-1 protein expressed on the membrane of CAR-T cells using CRISPR-Cas9 knock-in technology. The specific steps are as follows:

[0094] S800: Using CRISPR-Cas9 to knock in the IN044 sequence into the PD1 gene in CT106 cells.

[0095] 6.1. Sequence Synthesis

[0096] The IN044 sequence was first synthesized, and the amplicon was purified by 40 cycles of PCR (polymerase chain cycle reaction) and Ampure magnetic beads.

[0097] 6.2. Prepare gRNA-PD1

[0098] Design and synthesize gRNA-PD1 targeting the extracellular region of human PD-1 protein, resuspend crRNA and tracrRNA in IDT duplex buffer at a concentration of 200uM. The crRNA and tracrRNA were thawed and dissolved, mixed at a volume of 1:1, incubated at 98°C for 5 min, and cooled at room temperature to form a 100 μM gRNA-PD1 solution.

[0099] 6.3. Electroporation process

[0100] Day 0: Isolate T cells with magnetic beads,...

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Abstract

The invention relates to a gene biological preparation as well as a preparation method and application thereof, a section of IN044 sequence is knocked into a protein gene of the gene biological preparation by using a gene editing technology, so that a target gene in the protein gene is silenced, and a gene knockout effect is achieved. After the target gene is edited by using the method disclosed by the invention, on one hand, the frame-shift mutation target gene cannot be translated into the protein, and on the other hand, the generated protein can be quickly degraded, so that the toxicity generated by the translated protein with unknown characters is completely eradicated. The method is wide in application prospect and can be applied to various aspects such as gene editing, disease diagnosis and treatment and the like.

Description

technical field [0001] The invention relates to the fields of gene editing and biological cell technology, in particular to a gene biological preparation and its preparation method and application. Background technique [0002] In recent years, gene editing technology has developed rapidly, especially CRISPR-cas9, CRISPR (Clustered regularly interspaced short palindromic repeats) regularly clustered interspaced short palindromic repeats; Cas9 (CRISPR associated nuclease) is a CRISPR-related nuclease, and CRISPR-Cas9 is the latest emergence An RNA-guided technology that utilizes Cas9 nuclease to edit targeted genes. Two articles published in "Science" (Science) on February 15, 2013, proved that the Cas9 system can perform effective targeted enzyme digestion in 293T, K562, iPS and other cells, and non-homologous recombination (NHEJ ) and homologous recombination (HR) have respective efficiencies between 3-25%, and the recombination efficiency is equivalent to the cutting effe...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/113C12N15/11A61K48/00A61P35/00
CPCC12N15/85C12N15/113C07K14/00A61K48/005A61P35/00C12N2310/20
Inventor 谢海涛马丽雅都晓龙王乃会
Owner SHENZHEN FIRST CONDOR BIOSCIENCE CO LTD
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