Adipocyte-targeting non-viral gene delivery complex comprising dual plasmid vector

一种基因递送、脂肪细胞的技术，应用在使用载体引入外来遗传物质、载体、基因治疗等方向，能够解决未观察到体重减少效果等问题，达到细胞毒性优秀的效果

Active Publication Date: 2019-06-04
IUCF HYU (IND UNIV COOP FOUND HANYANG UNIV)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, in the aforementioned Patent Document 1, an attempt was made to achieve an obesity treatment effect by inhibiting the expression of FABP4 in adipocytes after preparing a gene delivery complex including the shFABP4 gene, however, no sufficient effect was observed. weight loss effect

Method used

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  • Adipocyte-targeting non-viral gene delivery complex comprising dual plasmid vector
  • Adipocyte-targeting non-viral gene delivery complex comprising dual plasmid vector
  • Adipocyte-targeting non-viral gene delivery complex comprising dual plasmid vector

Examples

Experimental program
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Effect test

Embodiment 1

[0075] Example 1. Preparation of sh(FABP4+FABP5) double plasmid vector

[0076] To prepare the sh(FABP4+FABP5) dual plasmid vector, psiRNA-DUO (InvivoGen, USA) as a dual RNAPolIII cassette vector for the expression of two shRNAs was used, and the specific process was as follows.

[0077] The test tube containing the frozen plasmid was spun to precipitate the DNA. To obtain a 1 μg / μl plasmid solution, the obtained DNA was resuspended in 20 μl of sterilized water, and the resuspended plasmid was stored at -20°C. To increase yield, transfection into the resuspended E. coli (Escherichia coli) was performed for plasmid amplification.

[0078] Next, the psiRNA-DUO plasmid was treated with Bbs I (NEB, 2 units of enzyme / μg plasmid DNA) as a restriction enzyme, and the large fragment (3180bp) was eluted with 0.7% low-melting point agarose gel, then diluted and purified A 0.1 μg / μl solution of the DNA fragment was obtained (Gus box). And, use Acc 65I and Hind III (used with NEB enzy...

Embodiment 2

[0080] Example 2. The content ratio optimization of sh(FABP4+FABP5) plasmid and ATS-9R

[0081] In order to optimize the content ratio of sh(FABP4+FABP5) plasmid and ATS-9R, using gel retardation assay, 1 μg of DNA, deionized water, and ATS-9R were mixed at room temperature according to different weight ratios. Incubate for 30 minutes to prepare oligo-peptoplex. The average diameter and surface zeta potential of the oligopeptide complexes were measured using a Zetasizer-Nano ZS (Malven Instruments, UK) DLS. Figure 1a and 1b , shows the results of gel retardation analysis and the size of the complex according to the weight ratio of sh(FABP4+FABP5) plasmid to ATS-9R, refer to Figure 1b , the optimal size can be derived when the weight ratio of sh(FABP4+FABP5) plasmid:ATS-9R is 1:2.

Embodiment 3

[0082] Example 3. Confirmation of FABP4 and FABP5 expression in adipocytes transfected with gene delivery complexes according to the present invention amount

[0083] 3T3-L1 cells, which are mouse-derived adipocytes, were differentiated into adipocytes, and shFABP4+ATS9R, shFABP4+ATS9R, shFABP5+ATS9R, sh(FABP4 / FABP5) were scrambled in the differentiated adipocytes After +ATS9R treatment, only RNA was isolated from cells using RNeasy Lipid tissue Mini kit (Qiagen) after 48 hours, and cDNA was synthesized. Thereafter, FABP4, FABP5 mRNA levels relative to GAPDH as an endogenous control were measured by RT-PCR measurement using Cyber ​​premix EX taq RT-PCR Kit. Figure 2a and 2b shows the measurement results, refer to Figure 2a and 2b , in mature adipocytes tested in vitro, it was confirmed that the mRNA levels of FABP4 and FABP5 decreased when sh(FABP4 / FABP5)+ATS9R, which is a dual plasmid vector, was actually treated. Furthermore, when FABP4 was reduced, it was confirme...

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Abstract

The present invention relates to: an adipocyte-targeting non-viral gene delivery complex comprising a sh(FABP4 + FABP5) dual plasmid vector; and treatment for obesity and obesity-induced metabolic syndromes by using the same and, more particularly, to a gene delivery complex comprising: an adipocyte-targeting sequence; an arginine 9 (R9) peptide; and a dual plasmid vector comprising a gene for treatment of obesity and obesity-induced metabolic syndromes, wherein the gene for treatment of obesity and obesity-induced metabolic syndromes is a base sequence inhibiting the expression of a FABP4 gene and a FABP5 gene. According to the present invention, in order to treat obesity-related diseases, a dual plasmid vector capable of simultaneously inhibiting the FABP4 and FABP5 genes is produced, and since a gene delivery complex is provided by binding the vector to a predetermined delivery system specifically delivering the same to adipocytes, an excellent and non-toxic obesity treatment effecttargeting only adipocytes can be achieved.

Description

technical field [0001] The present invention relates to adipocyte-targeted non-viral gene delivery complexes comprising sh(FABP4+FABP5) dual plasmid vectors and treatment of obesity and obesity-induced metabolic syndrome utilizing the same. Background technique [0002] Many gene therapy approaches have been developed as an alternative to traditional protein therapy methods, however there are still issues to be solved, one of which is the need to achieve effective delivery through the plasma membrane (in animal cells) and the nuclear membrane with minimal cytotoxicity. Gene injection (influx). [0003] Gene therapy systems can be roughly divided into viral vector-mediated systems and non-viral vector-mediated systems. Viral vectors constructed using retroviruses or adenoviruses have the advantage of being able to efficiently transfect (transfection) into cells, however, they have immunogenicity problems in vivo and Intrinsic problems associated with genetic recombination. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113A61K48/00
CPCC12N15/113C12N15/85C12N2310/3513C12N2330/51C12N2320/31C12N2830/20C12N2830/00A61K31/7088A61P3/04C12N2310/14C12N2310/531A61K48/00C12N2810/85A61K48/0016A61K48/0025
Inventor 金龙熙金亨缜
Owner IUCF HYU (IND UNIV COOP FOUND HANYANG UNIV)
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