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Recombinant gene set for collaborative liver targeting delivery of low density lipoprotein receptor (LDLR),vector group, exosome, construction method and application

A technology of recombinant genes and construction methods, applied in the field of genetic engineering, can solve the problems of off-target, lack of effective means, weak targeting ability of exosomes, etc., to promote protein translation, treat hyperlipidemia, and increase liver targeting. Effect

Active Publication Date: 2021-08-13
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Exosomes are good carriers for protein and nucleic acid drugs. At present, there are still several bottlenecks in the use of exosomes to deliver nucleic acids for gene therapy: 1) The natural exosomes have weak targeting ability, which will lead to insufficient therapeutic effect. Increase the injection dose of exosomes, therefore, the targeting of exosomes needs to be improved; 2) How to efficiently load the target RNA into exosomes to improve the performance of exosomes, there is still a lack of effective means; 3) Off-target problems will restrict From exosome delivery to clinical transformation, how to achieve tissue specificity of drugs carried by exosomes needs to be further explored

Method used

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  • Recombinant gene set for collaborative liver targeting delivery of low density lipoprotein receptor (LDLR),vector group, exosome, construction method and application
  • Recombinant gene set for collaborative liver targeting delivery of low density lipoprotein receptor (LDLR),vector group, exosome, construction method and application
  • Recombinant gene set for collaborative liver targeting delivery of low density lipoprotein receptor (LDLR),vector group, exosome, construction method and application

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preparation example Construction

[0043]The preparation method of the exosome of the present invention is simple and easy.

[0044] The present invention loads Apoa1 protein on the surface of exosomes by means of genetic engineering, and the modified exosomes can target the liver; uses RNA binding protein to bind target RNA, assists RNA to enter exosomes, efficiently loads target RNA, and the modified exosomes Secretosomes, as a means of intercellular protein and nucleic acid information molecule transmission, can effectively achieve the purpose of interfering with endogenous proteins; when miR-122, which is highly expressed in the liver, binds to IRES, its conformation changes and protein translation is promoted. Inserting the IRES element into the 5' end of LDLR mRNA can realize the tissue specificity of nucleic acid drugs carried in exosomes. After the above modifications, an engineered exosome that targets the liver and efficiently loads hepatocyte-responsive mRNA can be produced, which has ideal targeting...

Embodiment 1

[0048] according to Figure 4 (Schematic diagram of the hypothesis) to conduct the experiment:

[0049] Construction of the recombinant expression vector set: the recombinant expression vector set was completed by GenScript Biotechnology Company;

[0050] (1) The first recombinant expression vector: Pme1 and BstBI are selected as restriction sites;

[0051] (2) From the 5' end to the 3' end, the IRES fragment (as shown in SEQ ID No.3), the LDLR (human9) fragment (as shown in SEQ ID No.5) and the MS2 fragment (as shown in SEQ ID No. .5) to obtain the first recombinant gene, the nucleotide sequence is as shown in SEQ ID No.1 in the sequence listing;

[0052] (3) The second recombinant expression vector: Pme1 and BstBI are selected as restriction sites;

[0053] (4) From the 5' end to the 3' end, the kozak fragment (as shown in SEQ ID No.6), the first CD63 fragment (as shown in SEQ ID No.7), and the Apoa1 fragment (as shown in SEQ ID No. 8), CD63 second fragment (as shown in ...

Embodiment 2

[0057] The obtained exosomes were subjected to qPCR to detect the level of LDLR mRNA in exosomes. For the experimental steps, refer to "In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR / dCas9" (PMID: 30517011). The result is as image 3 As shown, compared with the control group, Exo IRES -LDLR-MS2+Kozak-CD63-Apoa1-CD63-MCP The level of LDLR mRNA was significantly increased.

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Abstract

The invention relates to the field of gene engineering, in particular to a recombinant gene set for collaborative liver targeting delivery of low density lipoprotein receptor (LDLR), a vector group, an exosome, a construction method and application. The invention provides a recombinant gene set for collaborative liver targeting delivery of the LDLR. The recombinant gene set comprises a first recombinant gene and a second recombinant gene; the first recombinant gene comprises an internal ribosome entry site (IRES) fragment, an LDLR fragment and an MS2 fragment sequentially connected from a 5' end to a 3' end; and the second recombinant gene comprises a kozak fragment, a CD63 first fragment, an Apoa1 fragment, a CD63 second fragment and an MCP fragment sequentially connected from a 5' end to a 3' end. The liver targeting capacity of the recombinant gene set of the invention is remarkably improved, and the recombinant gene set has the advantages that a nucleic acid drug is efficiently loaded, the off-target rate is low, and meanwhile, can also effectively treat hyperlipidemia and atherosclerosis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant gene set, vector set, exosome, construction method and application of coordinating liver-targeted delivery of LDLR. Background technique [0002] Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease caused by mutations in the low-density lipoprotein (LDL) receptor gene. The biochemical indicators are characterized by extremely elevated levels of plasma total cholesterol and LDL cholesterol. Manifested as xanthoma and premature coronary heart disease. For the heterozygous type, the patient has a mutation in one of the homologous genes, which can only produce half of the receptors with normal structure and function, and the increase in plasma cholesterol will gradually appear with age. For this heterozygous type, drugs can be used to stimulate the expression of LDL receptors, thereby reducing plasma LDL levels. However, for the homozygous ty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/867C12N5/10A61K38/17A61K48/00A61P3/06A61P9/10
CPCC07K14/705C12N15/86A61K38/177A61K48/0058A61K48/0008A61P3/06A61P9/10C07K2319/01C12N2740/15043
Inventor 袁丽君李者龙杨国栋韦梦影王辰孙汶齐刘云楠
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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