Exosome delivery CasRx gene silencing AAV vector and construction method and application thereof

A gene silencing and exosome technology, applied in the field of genes to eliminate off-target effects and small size

Inactive Publication Date: 2021-06-25
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Recent studies have shown that there are differences between "gene silencing" and "heterochromatin". "Conformation

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  • Exosome delivery CasRx gene silencing AAV vector and construction method and application thereof
  • Exosome delivery CasRx gene silencing AAV vector and construction method and application thereof

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Embodiment Construction

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The embodiments of the present invention and all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0028] see figure 1 , 2 , the present invention provides a technical solution: an AAV vector for delivering CasRx gene silencing through exosomes: the AAV vector contains CasRx protein expression, and U6 promoter drives sgRNA expression.

[0029] A method for constructing an AAV vector for exosome delivery of CasRx gene silencing as described above: when the AAV vector is isolated from the conditioned medium of the production cell, AAV can be combined with the exosome exoAAV, and the exosome is used to wrap the AAV vector Efficient delivery of gene vectors can be achieved.

[0030] An application of the AAV vector delivered by exosomes as described a...

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Abstract

The invention discloses an exosome delivery CasRx gene silencing AAV vector. The AAV vector contains CasRx protein expression, and a U6 promoter drives sgRNA expression. The invention discloses a construction method of the exosome delivery CasRx gene silencing AAV vector. According to the construction method, when the AAV vector is separated from a conditioned medium for producing cells, AAV can be combined with exosome exoAAV, and efficient delivery of the gene vector can be realized by wrapping the AAV vector with the exosome. Compared with the prior art, the AAV vector has the beneficial effects that 1, the gene knockout efficiency is higher (greater than 90%); 2, compared with RNA interference (shRNA), the off-target effect is eliminated; 3, the AAV vector has the capability of targeting nuclear RNA (including non-coding RNA); 4, splicing is manipulated, wherein misconnection defects and targeted exon jumping are corrected; and 5, the size is small, a single vector AAV-CasRx can be realized, and RNA can be guided in vivo.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to an AAV vector for delivery of CasRx gene silencing by exosomes and its construction method and application. Background technique [0002] On March 16, 2018, a major achievement published in the journal "Cell" brought a big leap forward in RNA editing technology. Scientists from the Salk Institute in the United States expanded the RNA editing ability with a new CRISPR family enzyme, and Name this new system "CasRx." A key feature of the new system, in addition to its high efficiency and absence of apparent off-target effects, is its reliance on an enzyme that is physically smaller than in previous studies. This is crucial for RNA editing technology, which makes it easier for the editing tool to be packaged into a viral vector and enter cells for RNA editing. Hiroshi Nishimasu, a scientist at the University of Tokyo who was not involved in the study, said: "In this study, the resea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/113C12N9/22
CPCC12N9/22C12N15/113C12N15/86C12N2750/14143C12N2310/20
Inventor 梁宇杰段莉徐晓徐丽梅
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN
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