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Short interference ribonucleic acid as novel anti-tumor gene therapeutic medicine

An anti-tumor and drug technology, applied in the field of a group of new anti-tumor gene therapy drugs short interfering RNA, can solve the problems of decreased activity and difficult to judge the efficacy

Inactive Publication Date: 2005-11-02
MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The activity of the former decreases in a short period of time after being introduced into cells, and the curative effect is difficult to judge, while the stability and specificity of the latter need to be further confirmed and improved.

Method used

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  • Short interference ribonucleic acid as novel anti-tumor gene therapeutic medicine
  • Short interference ribonucleic acid as novel anti-tumor gene therapeutic medicine
  • Short interference ribonucleic acid as novel anti-tumor gene therapeutic medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0007] Example 1: Sequence design of siRNA

[0008] Analysis of the silencing efficiency of siRNA from the length of the siRNA and the length of the overhanging sequence shows that the most effective siRNA duplex is composed of a sense strand and an antisense strand of 21 nucleotides, and each strand has a 3' Two nucleotides are overhanging at both ends, and the middle 19 nucleotides are complementary paired (PNAS 2001; 98:14428-14433.). The two deoxyribonucleotides with overhanging protrusions are equally effective as ribonucleotides, but the former is cheaper to synthesize and can tolerate more nucleases, so the present invention selects the siRNA sequence with dTdT overhanging overhangs.

[0009] It is more efficient to select target regions from the 50-100 nucleotides downstream of the start codon of the complete cDNA sequence of a particular gene (Genes and Development 2001; 15: 188-200.). Since there are many regulatory protein binding sites in the 5' or 3' untranslated...

Embodiment 2

[0063] Example 2: Transfection of siRNA

[0064] The present invention uses LipofectAMINE TM 2000 liposomes (Life Technologies, product number: 11668-027, trademark Invitrogen, http: / / www.invitrogen.com) were used for transfection, and the silencing effect was measured 18-45 hours after transfection. All operations are in accordance with the 96-well plate operating procedures described in 11668-027.pdf file. The synthesized siRNA powder was dissolved in water to prepare a 4000 nM stock solution. 18-20 hours before transfection, experimental cells were inoculated into 96-well plates at 8000-10000 cells / well in 200 μl of antibiotic-free DMEM tissue culture medium containing 10% fetal bovine serum. Dilute 4000nM siRNA to 75μl of 120nM, 400nM, 1200nM, 2000nM, 4000nM with antibiotic-free and serum-free DMEM culture solution, respectively. In five other test tubes, take 2μl of liposomes and 73μl of antibiotic-free and serum-free DMEM for culture Mix the siRNA solution and the lip...

Embodiment 3

[0066] Example 3: Detecting the Cytotoxicity of siRNA to Tumor Cells by MTT Method

[0067] The cells transfected according to the method of the above-mentioned Example 2 were kept at 37 degrees, containing 5% CO 2 Incubate for about 45 hours in an incubator with air and 100% humidity. MTT was made into a 2 mg / ml solution with PBS buffer, 50 μl was added to each well, and incubated at 37 degrees for 4 hours to reduce MTT to Formazan. The supernatant was aspirated and 150 μl of DMSO (dimethyl sulfoxide) was added to dissolve Formazan. Measure the optical density OD of each well at 560 nm with a microplate reader (Model3550, Bio-Rad) 560 . The OD value of each test well is subtracted from the background OD value (complete medium plus MTT, no cells) or the OD value of the blank drug well (complete medium plus different dilutions of the test drug plus MTT, no cells), and each repeat The OD values ​​of the wells were taken as mean ± SD. The survival rate of the cells is repres...

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Abstract

By means of biological computer technology, one group of gene sequence relevant to human tumor is obtained from GenBank. Based on RNA interference technology, one group of siRNA capable of inducing RNA interference is designed. Certain amount of siRNA is synthesized chemically and used as novel efficient and specific antitumor medicine. MTT detection shows that siRNA has obvious cytotoxic effect on MCF-7, BEL 7402, KB, HT-29 and other tuomr cells. Western Blotting detection shows that siRNA can lower the expression level of specific protein by 80-90%. Morphological observation shows that siRNA can cause tumor cell produce corresponding morphological change and chromatin concentration. Compared with available technology, siRNA is one antitumor medicine with completely new mechanism, high efficiency and fast speed.

Description

Technical field: [0001] The present invention relates to the design and synthesis of a group of siRNA (short interfering ribonucleic acid) drugs and their application in tumor gene therapy. Background technique: [0002] Inhibiting the expression of tumorigenesis-related genes is an important idea in the research and development of new anti-tumor drugs. The RNA interference (RNAi) technology (Nature 1998; 391: 806-811), discovered in 1998, has become an effective tool to inhibit the expression of target genes. RNA interference is considered to be a physiological mechanism to defend against exogenous nucleic acid intrusion that is ubiquitous from lower organisms to higher organisms, and it can rapidly and specifically degrade target mRNA. Therefore, based on the RNA interference that exists in this organism itself, artificially design and synthesize specific siRNA targeting tumor-related genes as a precursor to induce RNA interference, so that it can effectively degrade tumo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/7088A61K48/00A61P35/00
Inventor 邵荣光刘铁刚殷勤伟张敏
Owner MEDICINE & BIOENG INST OF CHINESE ACAD OF MEDICAL SCI
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