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siRNA for inhibiting expression of CTGF gene, pharmaceutical composition containing siRNA and use of pharmaceutical composition

A gene expression and base technology, applied in the field of nucleic acid and pharmaceutical composition, can solve the problems of poor stability of siRNA activity, slow progress, slow progress of drugs, etc.

Pending Publication Date: 2020-07-07
SUZHOU RIBO LIFE SCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the clinical application of siRNA drugs for the treatment of diseases related to CTGF gene expression has been slow. Among them, the activity of siRNA itself and its poor stability in blood are one of the reasons for the slow progress of this type of drugs.

Method used

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  • siRNA for inhibiting expression of CTGF gene, pharmaceutical composition containing siRNA and use of pharmaceutical composition
  • siRNA for inhibiting expression of CTGF gene, pharmaceutical composition containing siRNA and use of pharmaceutical composition
  • siRNA for inhibiting expression of CTGF gene, pharmaceutical composition containing siRNA and use of pharmaceutical composition

Examples

Experimental program
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Effect test

Embodiment

[0236] Unless otherwise specified, the reagents and media used in the following examples are commercially available, and the nucleic acid electrophoresis, real-time PCR and other operations used are all referring to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)) conduct.

[0237] Hela cells were provided by the Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, using 20% ​​fetal bovine serum (FBS, Hyclone Company) and 0.2 volume% penicillin-streptomycin (Penicillin-Streptomycin, Gibco, Invitrogen Company) Cells were cultured in complete DMEM medium (Hyclone Company) at 37 °C in 5% CO 2 / 95% air incubator.

[0238] Unless otherwise stated, the proportions of reagents provided below are all calculated by volume ratio (v / v).

preparation example 1

[0239] Preparation example 1 Synthetic siRNA sequence

[0240] In siRNA synthesis, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to the modified nucleoside phosphoramidite used in the solid-phase synthesis of phosphoramidite according to the type and sequence of nucleotides in the siRNA to be prepared Monomers (modified RNA phosphoramidites). Phosphoramidite solid phase synthesis is a method used in RNA synthesis well known to those skilled in the art. Unless otherwise specified, the nucleoside monomers used are commercially available.

[0241] (1-a) Solid phase phosphoramidite method

[0242] The siRNA sequences listed in Table 2 were obtained by the solid-phase phosphoramidite method.

[0243] For the sense strand, use a universal solid phase carrier (UnyLinker TM loaded HL SolidSupports (Kinovate Life Sciences company) starts the cycle, and connects nucleoside monomers one by one from the 3'-5' direction according to the sequence of nu...

experiment example 1

[0283] Experimental example 1 Detection of the inhibitory efficiency of siRNA on CTGF mRNA expression in Hela cells.

[0284] Using Lipofectamine TM 2000 The siRNA obtained in Preparation Example 1 was transfected into Hela cells respectively, and the final concentration of siRNA was 50 nM. Each siRNA was transfected in triplicate wells. Cells without any siRNA treatment served as blank control.

[0285] The expression levels of CTGF mRNA in Hela cells transfected with each siRNA were detected by real-time fluorescent quantitative PCR (Quantitative Real-Time PCR). The specific steps are: after culturing the transfected cells for 24 hours, use Trizol (Thermo Fisher Company) to extract the total RNA in the cells according to the standard operation procedure of total RNA extraction; take 1 μg of total RNA respectively, and use a reverse transcription kit (Promega Company) , Cat. No. A3500) were reverse-transcribed to obtain cDNA according to the operation method in the manual....

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PUM

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Abstract

The invention relates to siRNA for inhibiting expression of a CTGF gene. The siRNA contains a positive-sense strand and an antisense strand, wherein the positive-sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are both 19 nucleotides in length, and the nucleotide sequence II at least partially and mutually complements a sequence which is 19 nucleotides in length in a region from site No. 700 to site No. 1050 of CTGF mRNA. From a terminal 5' to a terminal 3', nucleotides at sites No.7, No. 8 and No. 9 of the nucleotide sequence I are fluoro-modified nucleotides, and nucleotides at sites No. 2, No. 6, No. 14 and No. 16 of the nucleotide sequence II are fluoro-modified nucleotides.The invention further provides a pharmaceutical composition containing the siRNA. The siRNA and the pharmaceutical composition containing the siRNA can be used for treating or improving diseases relevant with the expression of the CTGF gene.

Description

technical field [0001] The disclosure relates to a nucleic acid capable of inhibiting CTGF gene expression and a pharmaceutical composition containing the nucleic acid, belonging to the field of nucleic acid pharmacy. The present disclosure also relates to the use of these nucleic acids and pharmaceutical compositions. Background technique [0002] Liver fibrosis refers to the excessive deposition of diffuse extracellular matrix (ECM) in the liver. It is a compensatory response in the tissue repair process secondary to various forms of chronic liver injury, and it is also the cause of chronic liver disease that develops into cirrhosis, liver cancer, etc. Severe fatal diseases must go through the pathological process, so anti-hepatic fibrosis has become the top priority in the treatment of chronic liver diseases. [0003] At present, the means of treating liver fibrosis are very limited, mainly including two aspects: one is to remove the pathogenic factors for the primary di...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P1/16A61P13/12A61P11/00A61P1/00A61P9/00A61P9/10A61P17/00A61P27/02
CPCC12N15/113A61K31/713A61P1/16A61P13/12A61P11/00A61P1/00A61P9/00A61P9/10A61P17/00A61P27/02C12N2310/14
Inventor 张鸿雁高山康代武郑书全
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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