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Double helical oligonucleotides interfering with mrna used as effective anticancer agent

An oligonucleotide and anticancer agent technology, applied in the field of using double helix oligonucleic acid interfering with mRNA as an effective anticancer agent, can solve problems such as inability to assist in predicting changes, expensive monoclonal antibodies, and the risk of large immune responses.

Inactive Publication Date: 2009-04-29
塞隆药商公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] Experiments on modified cells do not help predict what happens to natural, unmodified cells in tumors
The possible treatment with monoclonal antibodies carries a considerable risk of eliciting an immune response in the organism
In addition, monoclonal antibodies are quite expensive, and there is no guarantee that their manufacture will have a reproducible and invariant response in individual recipients because genetically modified organisms are used in the manufacturing process

Method used

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  • Double helical oligonucleotides interfering with mrna used as effective anticancer agent
  • Double helical oligonucleotides interfering with mrna used as effective anticancer agent
  • Double helical oligonucleotides interfering with mrna used as effective anticancer agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0192] Example 1. Anti-Wnt1 mRNA inhibits cell growth through designer siRNA

[0193] Cell proliferation of MCF-7 cells was measured 48 hours after treatment with a 50 nm siRNA sequence specific for the Wnt1 gene, and cell growth rate was determined using MTS assay. Growth of siRNA-treated cells compared to untreated cells (CTRL), cells treated with scrambling non-coding siRNA (SC siRNA), and cells treated with siControl TOX (siTOX) and Docetaxel (DOC) . SCsiRNA and siTOX are used to measure non-specific cell growth inhibition caused by nucleic acid chemistry or transfection agents, respectively, and to confirm transfection efficiency. figure 1 Values ​​shown indicate the proliferation ratio of non-transfected control cells. About 88% of non-coding siRNA (Non-coding siRNA) had almost no effect on cell proliferation and transfection efficiency in these experiments. Very few tested siRNA sequences showed a strong ability to reduce cell proliferation, in some instances more t...

Embodiment 2

[0194] Example 2. The designed siRNA is specific and effective in decreasing the amount of Wnt1 mRNA

[0195]Next, we measured the amount of mRNA in MCF-7 after treatment with siRNAs ranked by inhibition value. The reduction of mRNA is the most direct result of the effect of siRNA. We then determined whether transfection of MCF-7 cells with siRNA against Wnt1 mRNA resulted in a decrease in mRNA levels. Analysis was performed 48 hours after transfection. All mRNA isolation, transcription into cDNA, and real-time PCR were performed as described in the Materials and Methods above. After treating MCF-7 cells with the W15 sequence, we observed a 61% reduction in mRNA compared to untreated controls. This experiment was also controlled for the specificity of our sequence. Additionally, we performed a similar experiment with A549 cells to see if there was any response. Wnt1 is known not to be expressed in A549 cells (He et al. 2004). We observed that A549 cells treated with W15 ...

Embodiment 3

[0197] Example 3. siRNA specific to Wnt1 can trigger a reduction in the amount of protein

[0198] After transfection with anti-Wnt1 siRNA, Western blotting analysis (Western blotting analysis) of the amount of Wnt1 in MCF-7 cells was completed ( figure 2 a). After 48 hours of treatment with the W15 sequence, the amount of Wnt1 in the cells will not only decrease to a lesser extent, but also according to the importance of the control group to reduce the cells treated with the W13 sequence, the amount of Wnt1 in MCF-7 will be slightly reduced after the WP sequence treatment.

[0199] Western blot analysis showed that the amount of phosphorylated beta-catenin in MCF-7 cells increased after anti-Wnt1 siRNA treatment. We observed a correlation between the reduction in the amount of Wnt1 and the reduction in the amount of c-myc and cyclin D1 in MCF-7 cells treated with the W15 or W13 sequence. We did not find such a change after WP sequence treatment.

[0200] These data represe...

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Abstract

The present invention relates to the application of double-helical oligonucleotides (siRNA) interfering with the mRNA of gene involved in carcinogenesis, particularly the Wntl, Wnt2 or Her3 gene. Such oligonucleotides may be modified chemically, used in conjunction with viral and non-viral vectors such as lipid complexes. Such oligonucleotides exhibit unusual antiproliferative properties against tumour cells and may be used in anti-tumour treatment.

Description

technical field [0001] The present invention provides a new anti-cancer agent (anti-tumour agent) using double-stranded oligonucleotides to interfere with the mRNA of cancerogenesis genes, especially Wnt1, Wnt2 or Her3 genes. Background technique [0002] RNA interference (RNA interference), a phenomenon based on post-transcriptional gene silencing (PTGS), is also an excellent tool for analyzing the function and role of many pathways in organisms. This technology is very important in functional genomics, corresponding biochemical pathways, determining the direction of drug therapy, and gene therapy. Post-transcriptional gene silencing (PTGS) was first described in plants (Napoli, C, C. Lemieux and R. Jorgensen. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2:279-289, 1990). In 1998, two people including Andrew Fire and Craig Mello first described RNA interference (RNAi) on animal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11G06F19/00C12N15/113G16B20/00
CPCC12N2320/11C12N15/113C12N2310/14C12N15/111G06F19/18C12N2330/30G16B20/00A61P35/00
Inventor 梅西耶·维乔雷克乔安娜·威奇克安娜·内索威兹凯塔琪娜·琪克萨史卡帕特·珍·古珊达莫妮卡·莲帕斯卡·帕奇拜兹
Owner 塞隆药商公司