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Asymmetric interfering RNA compositions that silence K-RAS and methods of uses thereof

A technology of gene silencing and nucleotides, applied in the field of compositions for silencing K-Ras gene expression, can solve problems such as the unknown role of oncogenes

Inactive Publication Date: 2017-12-01
1GLOBE BIOMEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of oncogenes such as K-Ras in cancer cell stemness is unknown

Method used

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  • Asymmetric interfering RNA compositions that silence K-RAS and methods of uses thereof
  • Asymmetric interfering RNA compositions that silence K-RAS and methods of uses thereof
  • Asymmetric interfering RNA compositions that silence K-RAS and methods of uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Example 1: In vitro potency of aiK-Ras

[0140] Figure 1(A) shows in vitro studies using aiRNA ID NO: 21 ("aiK-Ras#1") targeting the K-Ras target SEQ ID NO: 22 to determine the IC of aiK-Ras#1 50 . DLD1 cells (ATCC) were transfected with aiK-Ras#1. Forty-eight hours after transfection, cells were collected and RNA was isolated. Determination of the IC of aiK-Ras#1 ​​by qPCR 50 . The remaining mRNAs were normalized to GAPDH expression levels. IC 50 It is 3.1pM, indicating that aiK-Ras#1 ​​efficiently silences K-Ras gene expression.

[0141] Figure 1(B) shows an in vitro study using aiRNA ID NO: 142 ("aiK-Ras#2") targeting the K-Ras target SEQ ID NO: 142 to determine the IC of aiK-Ras#2 50 . DLD1 cells were transfected with aiK-Ras#2. Forty-eight hours after transfection, cells were collected and RNA was isolated. Determination of the IC of aiK-Ras#2 by qPCR 50 . The remaining mRNAs were normalized to GAPDH expression levels. IC 50 It is 3.5pM, indicating ...

Embodiment 2

[0142] Example 2: Off-target effects of aiK-Ras reduction

[0143] Figure 2(A) shows detection of siRNA and aiRNA loaded in RISC by northern blot analysis. To analyze small RNA RISC loading, HEK293Flag-Ago2 stable cell lines were transfected with aiRNA or siRNA duplexes. Cells were lysed at indicated time points and co-immunoprecipitated with Flag antibody (Sigma, Catalog #F1804). The immunoprecipitate was washed, RNA was extracted from the complex with TRIZOL (Life Technologies, 15596-018), and loaded onto 15% TBE-Urea PAGE or 15% TBE non-denaturing PAGE gel. After electrophoresis, transfer RNA to Hybonad-XL Nylon membrane. The r-P32-labeled probe for detecting the sense or antisense strand is then hybridized to the RNA on the membrane. HEK293 cells expressing Flag-Ago2 (Invivogen, Catalog#293-null) were transfected with siRNA or aiRNA, and then co-immunoprecipitation assay was performed. FLAG-Ago2 HEK293 cells stably expressing FLAG-Ago2 were formed by transient transf...

Embodiment 3

[0146] Example 3: aiK-Ras sensitivity in K-Ras mutant cells

[0147] Fig. 3(A) shows the colony formation assay of AGS (ATCC) and DLD1 transfected with aiK-Ras#1 ​​or aiK-Ras#2. Cells were transfected with 1 nM GFP aiRNA (control; GGTTATGTACAGGAACGCA (SEQ ID NO: 956)) or 1 nM aiK-Ras#1 ​​or aiK-Ras#2 for 24 hours. Cells were then trypsinized and transferred to 6-well plates at 500-2000 cells / well to test the clonogenicity of the cells. After 11-14 days, colonies were stained with Giemsa stain and counted. For western blot analysis, the cells were washed with ice-cold PBS, in lysis buffer [50mM Hepes (pH 7.5), 1% Nonidet P-40, 150mM NaCl, 1mM EDTA, 1X Halt Proteasome Inhibitor Cocktail (Thermo Scientific, Catalog #87786)]. Solubilized proteins (10 μg) were separated by SDS / PAGE and transferred to PVDF membranes. Primary antibodies were used in this assay. Antigen-antibody complexes were visualized by enhanced chemiluminescence (BioRad, Catalog #170-5060).

[0148] Figure ...

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Abstract

The invention provides novel compositions for use in silencing K-Ras gene expression. More particularly, the invention provides novel asymmetrical interfering RNA molecules as inhibitors of K-Ras expression, and to pharmaceutical compositions and uses thereof in the treatment of cancer or a related disorder in a mammal.

Description

technical field [0001] The present invention generally relates to compositions for silencing K-Ras gene expression. More specifically, the present invention relates to a novel asymmetric interfering RNA molecule as an inhibitor of K-Ras expression, a pharmaceutical composition and its use in the treatment of mammalian cancer or related diseases. Background technique [0002] Gene silencing via RNAi (RNA interference) through the use of small or short interfering RNA (siRNA) has emerged as a therapeutic tool. However, in addition to outstanding delivery issues, the development of RNAi-based drugs faces challenges such as limited potency of siRNAs, non-specific effects of siRNAs, e.g., interferon-like responses and sense-strand-mediated off-target gene silencing, and interactions with Surprisingly high or prohibitive costs associated with siRNA synthesis. The gene silencing efficacy of siRNA is limited to about 50% or less for most genes in mammalian cells. The manufacture ...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H21/04C12N15/00
CPCA61P1/04A61P35/00A61P43/00C12N15/1135C12N2310/14A61K31/7105C12N2310/31C12Q1/6886C12Q2600/158G01N33/57446G01N2333/82G01N33/5748G01N2333/914
Inventor 李嘉强
Owner 1GLOBE BIOMEDICAL CO LTD
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