siRNA (small interference ribonucleic acid) for inhibiting DAAO (D amino acid oxidase) gene expression and medical application thereof

A gene expression and application technology, applied in the field of siRNA that inhibits DAAO gene expression and its medical application, can solve the problems that have not been reported in rats, and achieve the effects of low toxicity, inhibition of expression, and good stability

Inactive Publication Date: 2012-07-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the d...

Method used

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  • siRNA (small interference ribonucleic acid) for inhibiting DAAO (D amino acid oxidase) gene expression and medical application thereof
  • siRNA (small interference ribonucleic acid) for inhibiting DAAO (D amino acid oxidase) gene expression and medical application thereof
  • siRNA (small interference ribonucleic acid) for inhibiting DAAO (D amino acid oxidase) gene expression and medical application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, siRNA expression plasmid effectively inhibits the expression of DAAO gene in rat glioma cell C6

[0034] Construct the siRNA expression vector that inhibits the expression of rat DAAO gene, and detect the expression of DAAO in rat glioma cell C6.

[0035] 1. siRNA design

[0036] Taking the DAAO gene (GeneID: 114027) reading frame (CDS) region (NCBI number: NM_053626) as the target sequence, according to the usual experience of siRNA design, select from the 50th base of the CDS region, at www.promega .com / siRNADesigner / default.htm website designs siRNA online, a total of 53 candidate sequences appear, and potential feasible siRNA sequences are selected according to the following conditions:

[0037] (1) GC content 36%-52%;

[0038] (2) The third base of the Sense chain is A;

[0039] (3) The sixth base of the Sense chain is A;

[0040] (4) The 10th base of the Sense chain is U;

[0041] (5) The 13th base of the Sense chain is not G;

[0042] (6)...

Embodiment 2

[0071] Example 2. Intrathecal injection of DAAO gene siRNA inhibits formalin biphasic pain in rats

[0072] 1. Rat formalin pain model

[0073] Before inducing pain, the rats were familiar with the environment in the transparent observation cage for about 30 minutes. After the initial exploration state became resting state, the rats were respectively placed in special cloth bags, and 5% Formosan was subcutaneously injected on the dorsum of the right hind foot of the rats. Malin 50μl, immediately after the injection, the rats were taken out of the cloth bag and put into a transparent observation cage for observation.

[0074] Observe once every 10 minutes, use a counter to count and record the number of times the rats are injected with the feet and legs within 1 minute (the number of spontaneous retraction or shaking), that is, record 0-1, 10-11, 20-21 after injection of formalin, 30-31, 40-41, 50-51, 60-61, 70-71, 80-81, 90-91 min the number of times the rats were inj...

Embodiment 3

[0115] Example 3, the use of DAAO siRNA in the preparation of drugs for the treatment of human chronic pain and schizophrenia

[0116] The mechanism of siRNA gene silencing is as follows Image 6 shown. RNAi and post-transcriptional gene silencing (post-transcriptional gene silencing and transgene silencing) have been proved to be the same phenomenon at the molecular level. A very important enzyme in RNA interference is Dicer of the RNaseIII ribozyme family. It can combine with double-stranded RNA, and cut it into 21-23nt and 3' protruding small molecule RNA fragments, that is, siRNA.

[0117] Then siRNA is combined with several proteins, called RNA-induced silencing complex (RISC) caused by RNA, unwinds into a single strand, and the complex dominates the RNAi effect (Caterina M J, Schumacher M A, Tominaga M, et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway [J]. Nature, 1997, 398:816-24).

[0118] After RISC is activated, the activated ...

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Abstract

The invention relates to siRNA (small interference ribonucleic acid) for inhibiting DAAO (D amino acid oxidase) gene expression and medical application thereof, belongs to the technical field of biology. The siRNA is selected from a sequence shown in a range from SEQ ID NO:1 to SEQ ID NO:10. The invention also relates to application of the siRNA in preparing a medicament for inhibiting DAAO gene expression, as well as a polycation composite and lipid containing the siRNA, as well as recombinant plasmid rhoDC316-EGFP (enhanced green fluorescent protein)-U6/shRNA (short hairpin ribonucleic acid) producing the siRNA. The siRNA can effectively inhibit expression of DAAO, has over 60 percent of analgesic effect on rat formalin second-phase pain by virtue of intrathecal injection. The siRNA and the expression plasmid thereof disclosed by the invention have the characteristics of strong specificity, high efficiency, high stability and low toxicity, can effectively inhibit expression of the gene DAAO, and can be applied to preparation of DAAO gene expression inhibiting medicaments, analgesic medicaments, schizophrenia medicaments and the like.

Description

technical field [0001] The invention relates to a small molecule interfering ribonucleic acid (siRNA) in the field of biotechnology and its medical application. [0002] Specifically, it relates to an siRNA that inhibits the expression of D-type amino acid oxidase gene (DAAO gene) and its medical application. Background technique [0003] Pain is an unpleasant but unavoidable reaction, which can make people avoid serious injuries, but long-term pain will become a serious burden on people's lives, and pathological pain is the cause of quite a lot of patients' suffering. Chronic pain is one of the most common and least treatable diseases in humans. According to statistics, 320 million people suffer from chronic pain every year, and at least 40% to 50% of chronic pain patients lack effective treatment. The literature "P. Ganju, J. Hall, Potential applications of siRNA for pain therapy, Exp. Opin. Biol. Ther. 4 (2004) 531–542" discloses the following content: Although there is...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63C12N7/01A61K48/00A61K9/127A61P25/00A61P25/18
Inventor 王永祥李欣燕陈晓玲
Owner SHANGHAI JIAO TONG UNIV
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