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Mitochondrion-targeted gene editing complex, preparation method and application thereof and mitochondrion genome editing method

A gene editing and mitochondrial technology, applied in the biological field, can solve problems such as difficulty in restoring target gene function, inability to treat oxidative stress, and physiological function effects, achieving significant antioxidant capacity, improving oxidative stress levels, and highly targeted treatments. Effect

Active Publication Date: 2021-01-22
钟刚
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current treatment strategies for oxidative stress do not face up to or emphasize the role of changes in mitochondrial structure and function, but simply apply antioxidants, so that after conventional antioxidants enter cells, they will not selectively interact with intracellular Organelle binding inevitably forms a stable combination with some targets that do not require binding, which may affect normal physiological functions, not only cannot treat oxidative stress, but may also be harmful to cells
[0006] The main idea of ​​current mitochondrial gene therapy is to edit and correct the mutated gene, or to promote the transformation of heterogeneity, so as to reduce the ratio of mutant and wild-type genomes, so as to achieve therapeutic effects. The representative method is mitochondrial DNA base editing, which The biggest disadvantage of this method is that the labeling efficiency is very low, and it is difficult to restore the function of the target gene

Method used

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  • Mitochondrion-targeted gene editing complex, preparation method and application thereof and mitochondrion genome editing method
  • Mitochondrion-targeted gene editing complex, preparation method and application thereof and mitochondrion genome editing method
  • Mitochondrion-targeted gene editing complex, preparation method and application thereof and mitochondrion genome editing method

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Embodiment

[0048] 1. Synthesis of mitochondria-targeted cationic liposomes:

[0049] Cationic liposomes are used to synthesize ordinary liposomes (Liposomes) with phospholipids (DOTAP) and cholesterol (Chol); the liposomes are modified with the classic mitochondrial targeting agent triphenylphosphine to endow the liposomes with mitochondrial targeting properties; On this basis, liposomes are covalently bonded with maleimide polyethylene glycol-FITC (MAL-PEG-FITC) micellar solution to increase its water solubility and stability, reduce immunogenicity, and simultaneously Give it a FITC fluorescent label.

[0050] 2. Construction and assembly of mitochondria-targeted gene editing complex (LCCT complex)

[0051] Use the region between 15953 and 15956 as the restriction site, and use U6 as the promoter to synthesize the corresponding crRNA;

[0052]

[0053] The double-stranded homologous template of TERT (dsDNA donor-TERT-mcherry) tagged with the reporter gene mcherry was constructed; t...

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Abstract

The invention provides a mitochondrion-targeted gene editing complex, a preparation method and application thereof and a mitochondrion genome editing method. The gene editing complex comprises a mitochondria-targeted cationic liposome and an mtCRISPR / Cas9 system whihc are a mitochondrial TERT gene double-stranded homologous template, a crRNA of a specific targeting TERT, an RNA homologous recombination repair template and a Cas9 protein. A gene editing technology based on CRISPR / Cas9 is combined with a mitochondrial targeting means, the mitochondrion-targeted gene editing complex is provided,a normal mitochondrial TERT gene functional fragment is introduced to replace a mismatched gene, and the oxidative stress level is improved by up-regulating a mitochondrial TERT gene.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mitochondria-targeted gene editing complex, a preparation method, an application, and a mitochondrial genome editing method. Background technique [0002] Osteoarthritis (OA) is a common joint disease in orthopedics, and its prevalence is increasing with the aging of the population. In recent years, more and more evidences have shown that oxidative stress is an important mechanism in the pathogenesis of osteoarthritis. Oxidative stress not only leads to cartilage destruction, but also participates in the secretion and degradation of inflammatory mediators, promoting the transition from clinically asymptomatic cartilage destruction to overt osteoarthritis. Oxidative stress is an imbalance between oxidation and anti-oxidation systems, which leads to a large accumulation of reactive oxygen species (Reactive oxygen species, ROS) in the body and excessive oxidation of cellular componen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/88C12N15/90C12N15/54C12N15/113C12N9/22A61K48/00A61K38/45A61K38/46A61P19/02
CPCC12N15/88C12N15/907C12N15/1137C12N9/22C12N9/1276A61K48/0008A61K48/005A61K38/45A61K38/465A61P19/02C12Y207/07049C12N2310/20
Inventor 钟刚
Owner 钟刚
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