siRNA for inhibiting adamts-5 gene and its application

A DNA molecule, methoxy modification technology, applied in the field of siRNA, can solve problems such as differences in silencing effects

Active Publication Date: 2018-01-09
ARGORNA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a variety of siRNAs can be designed for different fragment positions of the same gene, and the silencing effects are significantly different

Method used

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  • siRNA for inhibiting adamts-5 gene and its application
  • siRNA for inhibiting adamts-5 gene and its application
  • siRNA for inhibiting adamts-5 gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Embodiment 1, the screening of the effective oligomeric nucleic acid that suppresses ADAMTS-5 gene mRNA expression

[0155]1. Perform siRNA design to determine siRNA targeting ADAMTS-5, and perform bioinformatic screening to ensure that the sequence is specific to the ADAMTS-5 sequence and not specific to sequences from any other gene. The target sequence was checked against the sequence in GenBank using the BLAST search engine provided by NCBI. After preliminary experiments, 8 effective siRNAs were screened out, named siRNA-RB-01, siRNA-RB-02, siRNA-RB-03, siRNA - RB-04, siRNA-RB-05, siRNA-RB-06, siRNA-RB-07, siRNA-RB-08. The above siRNAs are siRNAs designed for different positions of the ADAMTS-5 gene sequence.

[0156] 2. Cell transfection

[0157] The experiment was divided into 10 groups, namely siRNA-RB-01 to siRNA-RB-08 experimental groups, No target (NTC) negative control group, and NC blank control group.

[0158] The setting method of siRNA-RB-01 to siRNA-R...

Embodiment 2

[0179] Embodiment 2, the inhibition of oligomeric nucleic acid to inflammatory factor

[0180] 1. The experiment is divided into the following groups:

[0181] hFLS-siRNA-RB-04 experimental group: primary culture hFLS cells to 6-well plates, when the cell density was about 50%, transfect hFLS cells with siRNA-RB-04 at a final concentration of 50nM according to the Lipofectamine2000 kit instructions.

[0182] 293T-siRNA-RB-04 experimental group: primary culture 293T cells to 6-well plates, when the cell density was about 50%, transfect cells with siRNA-RB-04 at a final concentration of 50nM according to the instructions of the Lipofectamine2000 kit.

[0183] hFLS-No target (NTC) negative control group: the siRNA in the hFLS-siRNA-RB-04 experimental group was replaced with random non-specific siRNA, and the rest of the steps were the same as in the hFLS-siRNA-RB-04 experimental group. Among them, the random non-specific siRNA is not specific to the siRNA designed for the target...

Embodiment 3

[0210] Embodiment 3, homologous oligomeric nucleic acid is to the verification of ADAMTS-5 gene inhibitory effect

[0211] In order to verify the influence of the homology ratio on the effect of siRNA-RB-04 on inhibiting ADAMTS-5 gene, the following three sets of experiments were carried out:

[0212] 1. The first set of experiments

[0213] The antisense strands of the first group of siRNAs are all "5'-AUGAUGCCCACAUAAAUCC-3'", and the sense strands are the homologous sequences of "5'-GGAUUUAUGUGGGCAUCAU-3'", as shown in Table 1.

[0214] Table 1 Antisense strand group

[0215]

[0216]

[0217] Note: S=sense strand, AS=antisense strand. Choose 15nt, 11nt, 23nt, 27nt, and mismatches for the sense strands, respectively.

[0218] According to the method of Example 1, each siRNA shown in Table 1 was transfected into hFLS cells, and its inhibitory efficiency on ADAMTS-5 gene mRNA expression was detected.

[0219] 2. The second set of experiments

[0220] The sense stran...

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Abstract

The invention discloses an siRNA for inhibiting ADAMTS-5 gene and an application of the siRNA. The invention discloses a chemically modified double-stranded siRNA molecule, which is a double-strandedsiRNA molecule formed by complementation of at least one strand of in the siRNA molecule shown in the following A after any chemical modification shown in the following (1)-(13). The siRNA disclosed by the invention can be used as a medicament for treating arthritis and related inflammations, and can be used for treating arthritis by locally injecting the siRNA and a preparation thereof into a bone articular cavity to inhibit expression of inflammatory factors.

Description

technical field [0001] The invention relates to siRNA for inhibiting ADAMTS-5 gene and application thereof, belonging to the field of biotechnology. Background technique [0002] Osteoarthritis (OA) is a chronic degenerative bone and joint disease that seriously endangers human health. At present, there is no effective treatment. There is an urgent need to develop new methods that can effectively prevent and treat osteoarthritis. One of the clinicopathological features of osteoarthritis is cartilage destruction and hFLS extracellular matrix degradation, and cartilage destruction is ultimately derived from the proteolysis of extracellular matrix. Therefore, the abnormal degradation of secondary extracellular matrix (ECM) caused by the increased activity of various proteolytic enzymes is the direct cause of cartilage degeneration, which eventually leads to the destruction of cartilage covering the surface of articular bone. Both interleukin-1 (IL-1) and tumor necrosis factor-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K31/713A61P29/00A61P19/02
CPCA61K31/713C12N15/113C12N2310/14C12N2310/315C12N2310/318C12N2310/321C12N2310/323C12N2310/3235C12N2310/3341C12N2310/335C12N2310/3513C12N2310/3515C12N2310/3517C12N2310/353
Inventor 张必良米其·托尔托雷王喆杨秀群王秋云
Owner ARGORNA PHARM CO LTD
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