Hypoxia response gene editing method

A gene editing and responsive technology, applied in the field of hypoxia-responsive gene editing, which can solve problems such as thermal damage

Pending Publication Date: 2021-05-28
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, photothermal therapy (PTT) utilizes thermal energy generated by photothermal materials to kill cancer cells, however, hig

Method used

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Experimental program
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Embodiment 1

[0023] A hypoxia-responsive gene editing method of the present invention comprises the following steps: mixing the hypoxia-responsive nanocomposite APACP with cells containing a target gene, the sequence of the target gene being: 5′-ATTCCCTGTCACCGCGTCAC-3′, p-carboxyl The specific reduction of azobenzene in the hypoxic microenvironment of the tumor leads to the breakage of the azo bond. Under hypoxic conditions, the Cas9 / sgRNA covalently linked to the hypoxia-responsive nanocomposite APACP is precisely and controllably released. And based on the CRISPR-Cas9 system, effective gene editing is realized, and tumor site treatment is realized under infrared light irradiation, and the target gene has the nucleotide sequence shown in SEQ ID NO.1.

[0024] The target genes include EGFP gene and HSP90α gene, the cells are A549 lung cancer cells, and the mice are BALB / c nude mice. In the tumor hypoxic microenvironment, the N-N double bond is destroyed, and the Cas9 / sgRNA complex is releas...

Embodiment 2

[0030] The preparation method of the hypoxia-responsive nanocomposite APACP of the present invention:

[0031] Preparation of gold nanorods AuNR5×10 by CTAB-mediated seed growth method-9 nmol, 0.0040g mercapto-polyethylene glycol-amino SH-PEG-NH 2 React with AuNR solution at 37°C for 12 hours to obtain SH-PEG-NH 2 -AuNR; 0.0036 g p-carboxyazobenzene activated with 0.0384 g N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide EDC and 0.0058 g N-hydroxysuccinimide NHS The carboxyl group of the carboxyl group was added for half an hour, then the p-carboxyazobenzene solution was added to the product obtained in the previous step, and incubated overnight at 37°C to form APA; at room temperature, in Tris-HCl buffer, 2.5 μg Cas9 protein was mixed with Incubate 7.5 μg sgRNA for 10 minutes to form Cas9 / sgRNA complex, mix activated APA with Cas9 / sgRNA complex, and store in a refrigerator at 4°C overnight, add PEI solution at a mass ratio of 5:1 to sgRNA The ratio of was transferred to the...

Embodiment 3

[0036] A hypoxia-responsive gene editing method of the present invention comprises the following steps:

[0037] Using CTAB-mediated seed growth method to prepare gold nanorods (AuNR), 0.0040g mercapto-polyethylene glycol-amino (SH-PEG-NH 2 ) reacted with AuNR solution at 37°C for 12 hours to obtain SH-PEG-NH 2 -AuNR; activation of the carboxyl group of p-carboxyazobenzene with N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and O-hydroxysuccinimide (NHS) After half an hour, the p-carboxyazobenzene solution was added to the product obtained in the previous step and incubated overnight at 37 °C to form APA. Cas9 protein was incubated with sgRNA at a Cas9:sgRNA weight ratio of 1:3 in Tris-HCl buffer (pH=7.4) for 10 min at room temperature to form a Cas9 / sgRNA complex. The activated APA was mixed with the Cas9 / sgRNA complex and stored in a refrigerator at 4 °C overnight. The PEI solution was transferred to the above product at a weight ratio of 5:1, and the final produc...

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Abstract

The invention discloses a gene editing method for hypoxia response. The nanometer composite material APACP is composed of Cas9/sgRNA and AuNRs modified by p-carboxyl azobenzene, and the outermost layer of the nanometer composite material APACP is wrapped with a layer of polyethyleneimine PEI. According to the present invention, 4-carboxylazobenzene is subjected to specific reduction in the tumor hypoxic microenvironment, such that the azo bond is broken, such that the accurate and controllable release of Cas9/sgRNA covalently linked to the APACP nano-particle is achieved, and the effective gene editing is achieved based on the CRISPR-Cas9 system; according to the photo-thermal conversion characteristic of AuNRs, after a target gene in tumor tissue is knocked out in Cas9/sgHSP90, a photo-thermal therapy is used for treating tumors. Cell and animal experiments prove that the treatment method is effective and superior to AuNRs photo-thermal treatment, and the wide potential of the APACP nanoparticles in tumor treatment is also proved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene editing method for hypoxia response. Background technique [0002] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) technology is currently the most popular gene editing tool, which plays a vital role in gene editing and disease treatment. So far, various methods have been developed to deliver the CRISPR-Cas9 system. However, there are still many challenges in how to efficiently and controllably deliver the CRISPR-Cas9 system. Hypoxia is a common feature in the microenvironment of most solid tumors. Considering the hypoxic microenvironment of tumors and the hypoxia responsiveness of azo bonds, it is possible to solve the problem of effective tumor targeting and controllable delivery of CRISPR-Cas9 technology. On the other hand, photothermal therapy (PTT) utilizes thermal energy generated by photothermal materials to kill cancer c...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K41/00A61K47/60A61K9/51A61K31/7088A61K38/46A61K47/32A61P35/00B82Y5/00
CPCA61K41/0052A61K48/0058A61K48/0008A61K38/465A61K9/5138A61K31/7088A61K47/60A61P35/00B82Y5/00A61K2300/00
Inventor 宋玉君李雪晴
Owner NANJING UNIV
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