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Composition for suppressing target gene expression

A composition and target gene technology, applied in gene therapy, drug combination, active ingredients of heterocyclic compounds, etc., can solve the problems of reduced efficacy, off-target, inability to effectively inhibit target genes, etc., to achieve the effect of improving efficacy and reducing off-target

Active Publication Date: 2010-03-03
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in typical experiments, the lowest possible concentration will reduce potency and affect the results of loss-of-function screens
[0004] In addition, people have also developed many compositions containing siRNA to inhibit the expression of target genes as drugs. These drugs generally contain siRNA that can specifically inhibit the expression of target genes. However, as mentioned above, siRNA usually has low efficacy in mammals. and a large number of off-target problems, therefore, cannot effectively inhibit the expression of target genes

Method used

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  • Composition for suppressing target gene expression
  • Composition for suppressing target gene expression
  • Composition for suppressing target gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] This example was used to determine the activity of siRNA alone and in combination with fluoroquinolones.

[0066] (1) Cell culture and transfection

[0067] Human embryonic kidney cells (HEK-293) were incubated at 37°C and 5% CO 2 They were cultured in DMEM / HIGH GLUCOSE (GIBICO) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin and 4 mM L-glutamic acid. Cells are maintained in exponential growth. Improved scheme (S.M.Elbashir, J.Harborth, W.Lendeckel, A.Yalcin, K.Weber, T.Tuschl, Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells, Nature.411(2001) 494 by reported -498.), transfection of two luciferase plasmids. After transfection, cells were seeded into 24-well plates (0.5 ml medium / well) to reach confluence (approximately 50%) at the time of transfection. After culturing for 24 hours, the medium was replaced with OPTIMEM 1 (GIBICO) (0.5 ml / well) before transfection. Cells were co-transfected...

Embodiment 2

[0074] This example is to illustrate the relationship between the concentration of enoxacin and the improvement of siRNA activity.

[0075] Using different concentrations of enoxacin, HEK-293 was cultured and transfected according to the same method as in Example 1, and then the activity of luciferase was measured. The activity improvement rate of siRNA produced by 100 μM enoxacin was set at 100%.

[0076] from figure 2 It can be seen that the 50% effective concentration (EC50) of enoxacin is about 30 [mu]M.

Embodiment 3

[0078] This example was used to determine the effect of enoxacin alone on firefly luciferase.

[0079] HEK-293 was cultivated and transfected in the same manner as in Example 1, except that only plasmids were used instead of siRNA.

[0080] Then, according to the same method as in Example 1, the cells were treated with different concentrations of enoxacin, and the activity of luciferase was determined. Inhibition by enoxacin was expressed as a normalized ratio between the luciferase reporter gene and the luciferase control gene. Luciferase control gene refers to samples not treated with the chemicals. The result is as image 3 shown.

[0081] from image 3 It can be seen that only at 120 μM and 150 μM, enoxacin inhibits the activity of luciferase, and has no effect on the activity of luciferase at concentrations lower than 120 μM. However, as measured in Example 2, the 50% effective concentration (EC50) of enoxacin is only about 30 μM. Therefore, within the effective concen...

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Abstract

The invention relates to a composition for suppressing target gene expression, which contains siRNA as an active component that can specifically suppress the target gene expression. The composition also comprises fluoroquinolone, wherein the mol ratio of the siRNA and the fluoroquinolone is from 1:5*10<2> to 1:1*10<7>. The composition for suppressing target gene expression comprises the siRNA andthe fluoroquinolone, the fluoroquinolone can increase the efficiency of the siRNA, disclosed as a picture 3, the effective concentration of 50 percent (EC50) of enoxacin is about 30 micrometers, and therefore, the composition can more effectively suppress the target gene expression. Compared with the singly used siRNA, the composition can realize the needed suppression effect under lower siRNA concentration, thereby reducing to miss targets; moreover, in an effective concentration range, the fluoroquinolone can not influence the normal functions of cells and organisms, and therefore, the composition is safe.

Description

technical field [0001] The present invention relates to a composition, more specifically to a composition for inhibiting target gene expression. Background technique [0002] RNA interference (RNA interference, RNAi) is a process in which double-stranded RNA (double-stranded RNA, dsRNA) molecules shut down the expression of the corresponding gene or silence the gene at the mRNA level. RNA interference technology, also known as gene knockdown (knock-down) or gene silencing (gene silencing), is a typical post-transcriptional gene regulation method, also known as post-transcriptional gene silencing (PTGS). ). RNAi was first elucidated by Fire and Mello in Caenorhabditis elegans (A.Fire, S.Xu, M.K.Montgomery, S.A.Kostas, S.E.Driver, C.C.Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature.391 (1998) 806-811.), RNAi is a conserved mechanism for RNA-guided regulation of gene expression in most eukaryotic organisms, in which dou...

Claims

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Application Information

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IPC IPC(8): A61K31/7105A61K31/47A61K31/496A61K31/5383A61K48/00A61P43/00
Inventor 席真张强哲张彩红
Owner NANKAI UNIV
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