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SiRNA capable of inhibiting expression of TIMP-1 gene, pharmaceutical composition containing siRNA, and applications of SiRNA and pharmaceutical composition

A TIMP-1, gene expression technology, applied in drug combinations, medical preparations containing active ingredients, DNA/RNA fragments, etc., can solve the problems of poor stability of siRNA activity, slow drug progress, and slow clinical application progress.

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

[0005] However, so far, the clinical application of siRNA drugs for the treatment of diseases related to TIMP-1 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. one

Method used

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  • SiRNA capable of inhibiting expression of TIMP-1 gene, pharmaceutical composition containing siRNA, and applications of SiRNA and pharmaceutical composition
  • SiRNA capable of inhibiting expression of TIMP-1 gene, pharmaceutical composition containing siRNA, and applications of SiRNA and pharmaceutical composition
  • SiRNA capable of inhibiting expression of TIMP-1 gene, pharmaceutical composition containing siRNA, and applications of SiRNA and pharmaceutical composition

Examples

Experimental program
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Embodiment

[0235] 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.

[0236] 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.

[0237] Unless otherwise stated, the proportions of reagents provided below are all calculated by volume ratio (v / v).

preparation example 1

[0238] Preparation example 1 Synthetic siRNA sequence

[0239] 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.

[0240] (1-a) Solid phase phosphoramidite method

[0241] The siRNA sequences listed in Table 2 were obtained by the solid-phase phosphoramidite method.

[0242] For the sense strand, use a universal solid phase carrier (UnyLinkerTMloaded 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 nucle...

experiment example 1

[0282] Experimental example 1 Detection of the inhibitory efficiency of siRNA on TIMP-1 mRNA expression in Hela cells.

[0283] The siRNA obtained in Preparation Example 1 was respectively transfected into Hela cells using LipofectamineTM2000, 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.

[0284] The expression level of TIMP-1 mRNA in Hela cells transfected with each siRNA was 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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Abstract

The invention relates to siRNA capable of inhibiting expression of a TIMP-1 gene. The siRNA contains a positive-sense strand and an antisense strand, wherein the positive-sense strand comprises a nucleotide sequence I; the antisense strand comprises a nucleotide sequence II; the length of the nucleotide sequence I and the length of the nucleotide sequence II are both 19 nucleotides; and the nucleotide sequence II is at least partially complementary with the sequence of 19 continuous nucleotides in the 350th-700th region of TIMP-1 mRNA. According to the direction from the 5'tail end to the 3 'tail end, the nucleotides at the seventh, eighth and ninth positions of the nucleotide sequence I are fluoro-modified nucleotides, and the nucleotides at the second, sixth, fourteenth and sixteenth positions of the nucleotide sequence II are fluoro-modified nucleotides. The invention also provides a pharmaceutical composition containing the siRNA. The siRNA and the pharmaceutical composition containing the siRNA can be used for treating or improving diseases related to expression of the TIMP-1 gene.

Description

technical field [0001] The disclosure relates to a nucleic acid capable of inhibiting TIMP-1 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] Hepatic fibrosis is a compensatory response in the tissue repair process secondary to various forms of chronic liver injury, and it is also a necessary pathological process for the development of chronic liver diseases to severe fatal diseases such as cirrhosis and liver cancer. Therefore, anti-hepatic fibrosis has become a The top priority in the treatment of chronic liver disease. [0003] At present, the means of prevention and treatment of liver fibrosis are very limited, mainly including two aspects: one is to remove the pathogenic factors for the primary disease, such as anti-virus, alcohol abstinence, etc.; the other is ...

Claims

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Application Information

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IPC IPC(8): C12N15/113A61K31/713A61P1/00A61P1/16A61P9/00A61P11/00A61P13/12A61P17/00A61P17/02A61P27/06
CPCC12N15/113A61K31/713A61P1/00A61P1/16A61P9/00A61P11/00A61P13/12A61P17/00A61P17/02A61P27/06C12N2310/141
Inventor 张鸿雁高山康代武
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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