CRISPRi-mediated exosome for inhibiting scar formation as well as preparation method and application thereof

An exosome and scar technology, applied in the field of genetic engineering, can solve the problems of loss of normal skin elasticity and strength, and achieve the effect of reducing off-target phenomenon, strong specificity and avoiding poor effect.

Active Publication Date: 2021-09-03
珠海乐维再生医学科技有限公司
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Problems solved by technology

3. Due to the changes in the tissue structure at the scar, the elasticity and strength of the normal skin are lost
During wound regeneration, fibroblasts that do not express En1 will transform into fibroblasts that express En1, and fibroblasts that express En1 will promote wound repair but also lead to scarring.

Method used

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  • CRISPRi-mediated exosome for inhibiting scar formation as well as preparation method and application thereof
  • CRISPRi-mediated exosome for inhibiting scar formation as well as preparation method and application thereof
  • CRISPRi-mediated exosome for inhibiting scar formation as well as preparation method and application thereof

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preparation example Construction

[0069] The preparation method of the above-mentioned exosomes is as follows: cells with exocrine function are cultivated under conditions suitable for cell growth so that the dCas9-KRAB fusion protein and sgRNA enter the exosomes, and the exosomes are isolated. Among them, the cells are preferably HEK293T cells or MSC cells. Specifically, the gene editing system provided above can be preferably used in the present invention, introduced into cells and cultured, and the supernatant is separated to obtain exosomes that inhibit scar formation.

[0070] The exosomes can be directly used as active ingredients to prepare a repair agent for inhibiting scar formation, and the specific ingredients can be: 1-5w / v% exosomes, 10-20v / v% glycerin, 5-10v / v% Polyethylene glycol, 0.5-1w / v% hydroxypropyl chitosan, 0.1-1w / v% sorbic acid and 2.5-6w / v% hydroxyethylcellulose. Among them, "w / v" refers to the number of grams of a certain substance contained in every 100ml of restoration agent; "v / v%"...

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Abstract

The invention relates to the field of gene engineering, and particularly provides a CRISPRi-mediated exosome for inhibiting scar formation as well as a preparation method and application thereof. According to the sgRNA provided by the invention, the Engrailed-1 gene edition has strong specificity by designing a specific nucleotide sequence responsible for identifying a target fragment, the off-target phenomenon can be remarkably reduced during gene editing, and the En1 gene is accurately and efficiently modified in a targeted manner. Meanwhile, the En1 gene can be accurately and remarkably inhibited through a gene editing system provided by the invention by utilizing a CRISPRi technology and taking an exosome as a carrier, which avoids the problems of poor effect and high cost of a traditional scar repair technology, has an important meaning for inhibiting scar formation along with high safety.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a CRISPRi-mediated exosome that inhibits scar formation and its preparation method and application. Background technique [0002] Scars formed after skin injuries are also called scars. In daily life, encountering scalds, burns, mechanical scratches, surgical sutures, etc. will cause the skin to form scars. The essence of scar formation is that the normal tissue structure of the original skin is damaged, and a disordered repair form occurs during the repair process of the body. Normal uninjured skin differs from scars in the following ways. One, the scarred area of ​​skin lacks sebaceous glands, hair follicles, and other dermal appendages compared to normal skin. Second, normal skin presents a regular weave structure, while scars present dense extracellular matrix fibers. Third, due to the changes in the tissue structure at the scar, the elasticity and strength of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N5/10C12N15/55C12N9/22C12N15/85A61K48/00A61K47/46A61P17/02
CPCC12N15/113C12N9/22C12N15/85C12N5/0603C12N5/0686C12N5/0662C07K14/47A61K48/005A61K48/0008A61K47/46A61P17/02C12N2310/20
Inventor 黄国滨周红艳李爱群鲁浩龚春丽王子轩
Owner 珠海乐维再生医学科技有限公司
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