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siRNA capable of inhibiting expression of COL1A1 gene in human beings and animals and composition containing same, and application thereof

A technology of amine compounds and modifiers, applied in DNA/RNA fragments, drug combinations, gene therapy, etc., can solve the problems that hinder the development of siRNA drugs, poor siRNA activity, and slow progress in clinical application, so as to shorten the process of animal experiments, Improved developmental progression, significant mRNA repression effects

Active Publication Date: 2018-07-06
SUZHOU RIBO LIFE SCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, so far, the clinical application of siRNA drugs for the treatment of diseases related to CoLI gene expression has been slow, and the activity of siRNA itself is poor, which is one of the reasons for the slow progress of such drugs
In addition, there are species differences in the target nucleic acids of siRNA for different species, which hinders the development of siRNA drugs to a certain extent.

Method used

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  • siRNA capable of inhibiting expression of COL1A1 gene in human beings and animals and composition containing same, and application thereof
  • siRNA capable of inhibiting expression of COL1A1 gene in human beings and animals and composition containing same, and application thereof
  • siRNA capable of inhibiting expression of COL1A1 gene in human beings and animals and composition containing same, and application thereof

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Experimental program
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preparation Embodiment 1

[0120] The present invention uses COL1A1mRNA (Genebank registration number: (NM_000088.3) as a template for siRNA design to obtain 3 species-conserved siRNAs, whose sequence information is shown in Table 1. At the same time, set the sense strand nucleotide sequence such as SEQ ID NO.11 The siRNA whose antisense chain nucleotide sequence is shown as SEQ ID NO.12 is numbered NC. NC is an irrelevant sequence that has no corresponding target action site with COL1A1 mRNA, and serves as a negative control.

[0121] Table 1: 7 siRNAs targeting COL1A1

[0122]

[0123]

[0124] The above siRNAs have a high degree of homology among different species, specifically, siRNA Col1 completely matches the target sequence (GAAUGGAGAUGAUGGGGAA, SEQ ID No.21) of human (NM_000088.3) and mouse (NM_007742.4); There is one nucleotide mismatch in the target sequence (GAACGGAGAUGAUGGGGAA, SEQID No.22) of mouse (NM_053304.1) and rhesus monkey (XM_015119317.1), that is, the 4th nucleotide mismatch....

experiment Embodiment 1

[0127] This experimental example is used to detect the inhibitory rate of the siRNA obtained in Preparation Example 1 on the expression level of COL1A1 mRNA in vitro.

[0128] Human cervical cancer cell line (Hela) (purchased from ATCC, CCL-2 TM ) was inoculated and cultured in a 24-well plate with DMEM complete medium containing 10% fetal bovine serum, 2mM L-glutamine, 100U / ml penicillin, and 100mg / ml streptomycin, and the seeding density was 4×10 5 Cells / well, 0.5ml per well, culture overnight at 37°C.

[0129] The specific operation steps of cell transfection are as follows: Dilute 500ng of each siRNA sample synthesized in Example 1 into 50 μl of Opti-MEM serum-free medium, and simultaneously add 1 μl of Lipofectamine TM 2000 (Invitrogen Company) was diluted in 50 μl of Opti-MEM serum-free medium, and the above two solutions were incubated at room temperature for 5 minutes, and mixed evenly. After the mixed solution was allowed to stand at room temperature for 20 minute...

preparation Embodiment 2

[0142] Reasonable chemical modifications were performed on the siRNA CT with good activity and the negative control NC, and the modification information is shown in Table 4.

[0143] Table 4: Sequences of Modified siRNA Molecules

[0144]

[0145] Among them, the uppercase letters C, G, U, A and T represent the base composition of nucleotides; the lowercase letter d indicates that the nucleotide on the right side of the letter d is a deoxyribonucleotide; the lowercase letter m indicates that the letter m The ribose group of one nucleotide on the left is 2'-methoxyribose group; the lowercase letter f indicates that the ribose group of one nucleotide to the left of the letter f is 2'-fluororibose group; the lowercase letter s indicates that the phosphate group between the deoxyribonucleotides on both sides of the letter is a phosphorothioate group.

[0146]The sense and antisense strands of the siRNAs listed in Table 4 were obtained by conventional solid-phase synthesis meth...

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Abstract

The invention provides a siRNA molecule capable of inhibiting the expression of the COL1A1 gene in human beings and animals and a pharmaceutical composition, and application thereof. The siRNA molecule provided by the invention has long-acting effect in inhibiting the expression of the COL1A1 gene in human beings and animals, and can treat or improve hepatic fibrosis and hepatic diseases related to hepatic fibrosis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an siRNA, a pharmaceutical composition and applications thereof; specifically, to an siRNA molecule capable of inhibiting the expression of COL1A1 genes in humans and animals, a pharmaceutical composition containing the siRNA molecule and applications thereof. 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 pathoge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/713A61P1/16A61P13/12A61P11/00A61P1/00A61P9/00A61P9/10A61P17/00
CPCC12N15/113C12N2310/14C12N2320/30
Inventor 张鸿雁高山
Owner SUZHOU RIBO LIFE SCIENCE CO LTD
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