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Gene editing and delivering system as well as preparation method and application thereof

A gene editing and delivery system technology, applied in the field of medicine, can solve the problems of complex preparation process, high toxicity, low lysosome escape efficiency, etc., and achieve the effect of avoiding the risk of degradation

Pending Publication Date: 2022-05-10
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently disclosed non-viral vectors have a complicated preparation process and high toxicity; they mainly mediate intracellular delivery through endocytosis, but the low lysosomal escape efficiency makes the gene editing system easy to be degraded, only small Partially entrapped gene editing systems are able to function, resulting in overall inefficiency

Method used

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  • Gene editing and delivering system as well as preparation method and application thereof
  • Gene editing and delivering system as well as preparation method and application thereof
  • Gene editing and delivering system as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] The embodiment of the present application provides a liposome capable of achieving membrane fusion, which can be used as a shell component to form a gene nano-delivery system mediated by membrane fusion. Membrane fusion liposomes include cationic liposomes DOTAP, DMPC and DSPE-PEG, which are mixed in proportion to form liposomes by film hydration method. The specific preparation method includes the following steps:

[0061](1) Membrane fusion liposome film was prepared by film hydration method: DSPE-PEG, DOTAP and DMPC dissolved in chloroform according to 3.8:0:96.2 (0% DOTAP), 3.8:10:86.2 (10% DOTAP) , 3.8:20:76.2 (20% DOTAP), 3.8:30:66.2 (30% DOTAP), 3.8:40:56.2 (40% DOTAP) molar ratio mixed in the round bottom flask, chloroform volatilized to make it in the bottle The bottoms formed lipid films labeled 0% DOTAP, 10% DOTAP, 20% DOTAP, 30% DOTAP and 40% DOTAP.

[0062] (2) the pure ddH 2 O was added to the round-bottomed flask that formed the lipid film, oscillated a...

Embodiment 2

[0065] The embodiment of the present application provides the first gene nano-delivery system mediated by membrane fusion. The structure includes a membrane fusion liposome shell and a calcium carbonate precipitate hybrid gene plasmid core, and the membrane fusion liposome shell is wrapped in a calcium precipitate hybrid. The surface of the inner core of the gene editing plasmid; the membrane fusion liposome outer layer includes cationic liposome DOTAP, DMPC and DSPE-PEG; the preparation method of the calcium carbonate hybrid gene editing plasmid includes Ca 2+ , CO 3 2- Mixing co-precipitation with EGFP plasmid (reporter plasmid) to form a calcium carbonate hybrid plasmid core; the specific preparation method of the gene nano-delivery system includes the following steps:

[0066] (1) 16μLCaCl 2 (0.5M) solution, 2μg EGFP plasmid p1 and ddH 2 O is mixed to form 50 μL of LA solution, and B solution is composed of 16 μL of Na 2 CO 3 (0.01M) solution with ddH 2 O was mixed a...

Embodiment 3

[0073] The embodiment of the present application provides the second gene nano-delivery system mediated by membrane fusion, the structure includes a membrane fusion liposome whose structural lipid is DOPE and a calcium carbonate precipitated hybrid gene plasmid, and the membrane fusion liposome is wrapped in The surface of the calcium precipitation hybrid gene editing plasmid; the membrane fusion liposome outer layer includes cationic liposome DOTAP, DOTAP and DSPE-PEG; the preparation method of the calcium carbonate hybrid gene editing plasmid includes Ca 2+ , CO 3 2- Mixing co-precipitation with EGFP plasmid (reporter plasmid) to form the core calcium carbonate hybrid plasmid core; the specific preparation method of the gene nano-delivery system includes the following steps:

[0074] (1) 16μLCaCl 2 (0.5M) solution, 2μg EGFP plasmid p1 and ddH 2 O is mixed to form 50 μL of LA solution, and B solution is composed of 16 μL of Na 2 CO 3 (0.01M) solution with ddH 2 O was mi...

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Abstract

The invention belongs to the technical field of medicine, and particularly relates to a gene editing delivery system and a preparation method and application thereof. The gene editing nano-delivery system comprises a membrane fusion liposome outer layer and a calcium precipitation hybrid gene editing plasmid kernel, the outer layer of the membrane fusion liposome is coated on the surface of the inner core of the calcium precipitation hybrid gene editing plasmid. According to the invention, high-efficiency delivery of the gene editing system in cells is realized through membrane fusion mediation, an endocytosis pathway can be directly bypassed, so that the risk of lysosome degradation after endosome embedding is avoided, high-efficiency CRISPR / Cas9 plasmid delivery is expected to be realized, and the technical problem that a current gene editing system is difficult to deliver into the cells is effectively solved; through modification and transformation of the nano-carrier and a CRISPR / Cas9 system, precise gene editing treatment of liver related diseases such as hepatitis, liver cancer, hepatic failure and hepatic fibrosis can be realized.

Description

technical field [0001] The application belongs to the field of medical technology, and in particular relates to a gene editing delivery system and its preparation method and application. Background technique [0002] CRISPR / Cas9 gene editing technology can achieve precise targeted gene editing by editing the target gene sequence, and has been widely used to fight against viruses, bacteria, cancer, genetic diseases and other diseases that are closely related to human health. Currently, viral vectors are mainly used for gene editing, but they have the following limitations, mainly including: (1) CRISPR / Cas9 system (CRISPR / Cas9 plasmid ~ 10,000 bp) with low load, which is difficult to load; (2) immunogenicity, Inflammation, potential carcinogenicity; (3) low versatility; (4) lack of organ / cell targeting specificity. Non-viral nanocarriers can overcome the above-mentioned limitations of viral vectors and provide a carrier platform with promising clinical applications. However,...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/127A61K47/02A61K47/24A61P1/16A61P31/20B82Y5/00B82Y40/00
CPCA61K48/0058A61K48/0008A61K9/1271A61K47/02A61K47/24A61P1/16A61P31/20B82Y5/00B82Y40/00
Inventor 李明强陶玉孔慧敏卓陈雅易可郑春雄钟庆国
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV