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Modified siRNA and pharmaceutical composition containing the modified siRNA

An unmodified, -OR2OR1 technology, used in the field of RecQL1 gene expression inhibitors and cell death inducers, to solve problems such as reduced siRNARNAi activity

Active Publication Date: 2021-04-23
GENECARE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, modifications are known to generally result in reduced RNAi activity of siRNAs

Method used

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  • Modified siRNA and pharmaceutical composition containing the modified siRNA
  • Modified siRNA and pharmaceutical composition containing the modified siRNA
  • Modified siRNA and pharmaceutical composition containing the modified siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143]

[0144] The present inventors studied in vitro the RNAi activity of modified RecQL1-siRNAs containing 2'-substituted nucleotides at various positions using various cell lines.

[0145]

[0146]

[0147] Cells from the following human cell lines were used in the experiment: TOV-112D (ovarian cancer endometrioid adenocarcinoma), ES-2 and TOV-21G (ovarian cancer clear cell adenocarcinoma), HCT-15 (colorectal cancer), A549 ( lung cancer) and HeLa (cervical cancer) cells. Cell lines were obtained from ATCC (American Type Culture Collection, American Type Culture Collection). The cells of these cell lines were injected at 2.0×10 per well the day before siRNA transfection. 4 The amount of seeding in 24-well plate, or 2.0×10 per well 3 The amount of each was inoculated in a 96-well plate, and the following culture medium was used for culture: For TOV-112D, TOV-21G and HeLa, use DMEM (nacalai tesque) + 10% FBS (fetalbovine serum, fetal bovine serum, Sigma-Aldrich ) + 1...

Embodiment 2

[0166]

[0167] The present inventors investigated whether the modified RecQL1-siRNA (QL-19), which exhibited high RNAi activity in vitro in Example 1, also had an effect in vivo, using a tumor-bearing animal model.

[0168]

[0169]

[0170] TOV-112D cells (human ovarian cancer endometrioid adenocarcinoma, grade 3, stage IIIc, obtained from ATCC), in DMEM medium containing 10% FBS (Sigma-Aldrich) and 1% penicillin / streptomycin (nacalai tesque), subculture at a confluence of not more than 80% in an incubator at 37°C. Cells were washed with PBS, stripped and recovered with 0.05% trypsin-EDTA (nacalai tesque). The recovered cells were centrifuged at 300 xg, 3 minutes, 4°C. Suspend pelleted cells in cold PBS at a density of 2 x 10 7 cells / ml to obtain TOV-112D cell suspension.

[0171] 4-week-old female Balb / c nude mice (CAnN.Cg-Foxn1nu / CrlCrlj) were purchased from Charles River Corporation, Japan. After one week of acclimatization, 500 μl of the above TOV-112D cell sus...

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Abstract

The present invention provides a modified siRNA for inhibiting the expression of RecQL1 helicase gene, a RecQL1 gene expression inhibitor and cell death inducer containing the modified siRNA, and a drug combination containing the modified siRNA for treating cancer thing. Specifically, the present invention provides a modified siRNA as shown below, a RecQL1 gene expression inhibitor and a cell death inducer containing the modified siRNA, and a pharmaceutical composition for treating cancer containing the modified siRNA: The modified siRNA comprises a sense strand containing the nucleotide sequence shown in SEQ ID NO: 1 and an antisense strand containing the nucleotide sequence shown in SEQ ID NO: 2, and takes the RecQL1 helicase gene as a target double Chain-modified siRNA, wherein, the sense strand contains 2'-substituted nucleotides at positions 2, 3, 4, and 13 of the base sequence shown in SEQ ID NO:1, and the sense strand is selected from Contain 2'-substituted nucleotides at one or more positions in the group consisting of the 12th, 14th, 17th, 18th, and 19th positions of the base sequence shown in SEQ ID NO:1, and the 2'-substituted nucleotides The 2' position is ‑R 1 ,-OR 1 ,-R 2 OR 1 ,-OR 2 OR 1 or-R 3 OR 2 OR 1 , where R 1 for C 1‑4 Hydrocarbyl, R 2 and R 3 independently for C 1‑3 Alkylene.

Description

technical field [0001] The present invention relates to a modified siRNA for inhibiting the expression of RecQL1 helicase gene, a RecQL1 gene expression inhibitor and a cell death inducer containing the modified siRNA, and a drug combination containing the modified siRNA for treating cancer things. Background technique [0002] DNA helicase is an enzyme that unwinds double-stranded DNA into single strands, and plays an important role in various processes related to genetic information such as DNA replication, repair, transcription, translation, and recombination. There are many types of DNA helicases, and DNA helicases that show homology to the RecQ helicase of Escherichia coli are called RecQ-type helicases. [0003] There are five RecQ-type helicases (RecQL1, WRN, RTS, BLM, and RecQ5) in the human genome. Among them, variants in the WRN, RTS, and BLM genes are responsible for the genomic instability disorders of Werner syndrome, Rothmund-Thomson syndrome, and Bloom syndr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113A61K31/713A61K48/00A61P35/00A61P43/00
CPCA61K48/00A61K31/713C12N15/113A61P35/00A61K48/0016
Inventor 二见和伸古市泰宏金户聪
Owner GENECARE RES INST